1997-02-05 Assigned to HUGHES ELECTRONICS reassignment HUGHES ELECTRONICS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MEGERLE, CLIFFORD A.
A sensor system and method are provided that are capable of the real-time detection of target live microorganisms, such as biological warfare agents. The sensor system includes a highly-sensitive, highly-selective sensor cell that comprises a single-stranded oligonucleic acid sequence that is complementary to a portion of the DNA of a target live microorganism, the oligonucleic acid having been modified with the covalent attachment of electron donor and acceptor moieties. In the presence of the targeted microorganism, hybridization occurs between the modified oligonucleic acid and the microorganism's DNA, such that the electron conductance between the electron transfer moieties greatly increases, thereby providing a means of detecting the presence of the target live microorganism. Aside from the sensor cell, the sensor system also includes an inlet port in the sensor cell wall by which to introduce a sample from the fluid environment into the sensor cell; a cell wall disrupter to release the nucleic acid of the fluid sample into the sensor cell; an electron transfer rate measuring system to gauge the electron transfer rate between the electron transfer moieties of the modified oligonucleic acid; a power source; a microcontroller to analyze the measured electron transfer rate for evidence of hybridization; and a communication system for relaying information regarding the presence or absence of the target live microorganism to the user of the sensor system. It is contemplated that the sensor system, exclusive of a battery and pump pack, will be only slightly larger than a pack of cigarettes and light enough to be comfortably worn and carried by personnel.
The present invention relates generally to sensors for specific nucleic acid sequences. More particularly, the present invention relates to a real-time sensor system for the detection of target live agent deoxyribonucleic acid, such as would be useful in the detection of biological warfare agents.
The detection of bacteria and viruses that pose a threat to human populations is an invaluable capability. With early detection of a harmful microorganism, persons located in the vicinity of an infected area may be notified so that they might take necessary precautions for their protection, such as fleeing to a safe haven, consuming appropriate antibiotics, or donning protective gear.
While the proliferation of harmful air-borne or water-borne microorganisms in an area might be a natural occurrence or, at least, an unintended result of human interference (e.g., the contamination of bodies of water by raw sewage), the potential for rogue governments and terrorists to employ biological warfare agents (BWA's) against troops and civilian populations is an increasing concern that makes the need for sensor systems for harmful microorganisms all the more urgent. The sheer variety of BWA's that might be employed requires that a sensor system should be highly sensitive and highly selective with regard to target live agents. Examples of potential BWA's include anthrax, typhoid fever, smallpox, and valley fever. Given the speed with which BWA's might adversely affect a population, it would be highly desirable that the sensor system offer real-time detection.
There are available methods by which harmful microorganisms might be detected, such as antibody-based techniques and gene-probe assays. However, antibody-based techniques suffer from poor specificity and are not adaptable to field applications. Gene-probe assays have been considered a better alternative to antibody-based techniques alone, but gene-probe assays have been known to require numerous time-consuming steps which are difficult to automate. Neither antibody-based techniques nor traditional gene-probe assays offer the real-time sensor capabilities and the specificities needed to detect harmful microorganisms such as BWA's.
Recently, techniques have been developed which enable the highly specific and sensitive detection of live agents by addressing the genetic material of the biological warfare agent itself. These techniques are disclosed in (1) a patent application having International Publication Number WO 95/15971 (entitled "Nucleic Acid Mediated Electron Transfer"), naming Thomas J. Meade and Jon F. Kayyem as inventors and having been assigned on its face to California Institute of Technology; and (2) a publication entitled "Electron Transfer through DNA: Site-Specific Modification of Duplex DNA with Ruthenium Donors and Acceptors" (Angew. Chem. Int. Ed. Engl., Vol. 34, No. 3, pp. 352-354 (1995)), written by Thomas J. Meade and Jon F. Kayyem.
The method developed by Meade and Kayyem (hereinafter Meade et al) provides for the site-selective modification of nucleic acids with redox active moieties such as transition metal complexes. Specifically, Meade et al demonstrate the placement of ruthenium-containing electron donor and electron acceptor groups onto the ribose backbone of a single strand of DNA, allowing the placement of a ruthenium atom at or near each end of a single strand of DNA. The significance of this feat lies in the electrical conductance differential between ruthenium-doped single strands of DNA compared to that of a double helix comprising one such single strand and a non-doped complementary strand. In general, a double strand of polynucleotide is about one million times more electrically conductive than a single strand. When a ruthenium-doped single strand is combined with its non-doped complementary strand, one of the ruthenium-containing groups serves as an electron source and the other as an electron sink, such that electrons flow back and forth between these two groups. Thus, the double helix creates a highly conductive path along its molecular axis between the electron donor and the electron acceptor that does not exist in a single strand of DNA. In addition to serving as electrical connectors into and out of the molecule, ruthenium atoms have the added virtue of neither disrupting nor distorting the overall shape of the DNA backbone.
Prior to the method of Meade et al, the long range transference of electrons in a DNA matrix was hindered by such factors as (1) the random distribution and mobility of the electron donor and the electron acceptor pairs; (2) the potential short distances between the donor and acceptor; and (3) the loose and reversible association of donors and acceptors. Meade et al overcome these obstacles by teaching a method directed to the modification of nucleic acids with electron transfer moieties covalently attached to specific sites of nucleic acids on a single strand of DNA in a way that creates no stearic hindrance to hybridization to form a duplexed strand pair. The resulting doped DNA strand is capable of hybridizing to a complementary target sequence in a single stranded nucleic acid and thereafter rapidly transferring electrons between the donor and acceptor.
The method of Meade et al specifically involves the synthesis of two sets of complementary oligonucleotide strands modified first with a terminal aminoribose and then covalently modified with electron donor and acceptor moieties, such as redoxactive ruthenium complexes. Since the ruthenium complexes will react with the heterocyclic nitrogen atoms of the bases, the bases are protected by employing an unmodified complementary strand as a large hydrogen-bonded blocking group. After reaction with the ruthenium complex, the complementary strand is removed and the modified oligonucleotide is purified in accordance with known procedures.
By employing the methods of Meade et al, one may discriminate between DNA strands that are identical to the original and those that differ by, for example, one base pair out of fifteen pairs (i.e. a match of 14 of the 15 base pairs in a strand), or by more than one base pair, by observing the electrical conductivity of an oligonucleotide. Meade et al have thus developed an electrical method of distinguishing between different sequences of DNA. Meade et al expressly contemplate employing their method of DNA strand detection in the creation of bioconductors and diagnostic probes, such as for medical applications.
A need remains for a real-time sensor system for harmful microorganisms that offers highly sensitive and highly selective characteristics; that may be employed for environmental sampling in the field, such as air and water sampling; and that is capable of alerting both those in its immediate vicinity as well as those at remote locations of the presence and type of microorganisms detected. Further, the sensor should be conveniently sized for use in the field and should be relatively inexpensive to produce and operate.
(g) a communication system for relaying information regarding the presence or absence of the target sequence of nucleotides in the fluid to a user of the sensor system.
A sensor made in accordance with the present invention will alert the user to the presence of the target live microorganisms, thereby enabling the user to take protective actions such as withdrawing from the infected area, consuming appropriate antibiotics, and/or donning personal protective gear. For example, it is contemplated that the present sensor is particularly well-suited for use by troops, or in unattended ground sensors or in manned or unmanned ground vehicles or aircraft, to detect the presence of BWA's. Notably, if the present sensor system is linked to a communication network, it may be used to alert remote personnel to the threat of BWA's (such as battlefield command and control personnel) and to relay to remote personnel the location and type of BWA's present, such as by tandem use of a digital radio link and a global positioning satellite receiver. A field commander so alerted would then be able to adjust his tactics knowing the location and extent of the biological agent threat on the battlefield.
It is contemplated that a sensor system made in accordance with the invention will be only slightly larger than a pack of cigarettes and light enough to be comfortably worn and carried by personnel without difficulty throughout their daily activities, or to be mounted in very small ground sensors vehicles, or aircraft. It incorporates very low-cost technology and will have a field replaceable, disposable sensor cell.
FIG. 6 depicts a specific example of a ruthenium electron transfer donor moiety using bisbipyridine and imidazole as the ligands.
A sensor system is provided herein to detect airborne and waterborne target live microorganisms in real time. The sensor system offers highly specific and highly sensitive detection abilities using such techniques as developed by Thomas J. Meade and Jon F. Kayyem (hereinafter "Meade et al") and disclosed in the following two references: (1) a patent application having International Publication Number WO 95/15971 (entitled "Nucleic Acid Mediated Electron Transfer"), naming Thomas J. Meade and Jon F. Kayyem as inventors and having been assigned on its face to California Institute of Technology; and (2) a publication entitled "Electron Transfer through DNA: Site-Specific Modification of Duplex DNA with Ruthenium Donors and Acceptors" (Angewandte Chemie--International Edition in English, 34(3), pp. 352-354 (1995)), written by Thomas J. Meade and Jon F. Kayyem.
In short, Meade et al teach a method by which an oligonucleic sequence may be synthesized that is complementary to that of a targeted live microorganism and that has covalently attached electron acceptor and donor moieties at or near opposing ends of the sequence. The electron conductance of a single strand of such a doped oligonucleic sequence is roughly 100 electrons per second while, in comparison, a double helix exhibits an electrical conductance of about 1,000,000 electrons per second, or a 10,000-fold increase. When a ruthenium-doped single strand is combined with its non-doped complementary strand, one of the ruthenium-containing groups serves as an electron source and the other as an electron sink, such that electrons flow back and forth between these two groups. Thus, the double helix creates a highly conductive path along its molecular axis between the electron donor and the electron acceptor that does not exist in a single strand of DNA. The present sensor system employs this technology to detect target live microorganisms: a doped oligonucleic sequence is attached to an electrode in the sensor system and its electrical conductance is systematically measured. An electron conductance increase by several orders of magnitude indicates that the doped oligonucleic acid has hybridized with a complementary strand and that the target live microorganism is within the vicinity of the sensor system. While the present sensor system is not limited to the method by which Meade et al achieve a synthesized oligonucleic acid terminated with electron acceptor and donor moieties, no other method is presently known by which such site-selective modification of oligonucleic sequences may be achieved.
At the outset, it is noted that unless otherwise stated, the terms "nucleic acid" or "oligonucleotid" or grammatical equivalents thereof herein means at least two nucleotides covalently linked together. A nucleic acid employed in the present sensor system will generally contain phosphodiester bonds, although in some cases a nucleic acid may have an analogous backbone comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphos-phoroamideite linkages, or peptide nucleic acid linkages, as described in Meade et al, '971. The nucleic acids may be single stranded or double stranded, as specified. The nucleic acid may be DNA, RNA, or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribonucleotides, and any combination of uracil, adenine, thymine, cytosine, and guanine. In some instances, e.g., in the case of an "intervening nucleic acid", the term nucleic acid refers to one or more nucleotides.
The terms "electron donor moiety", "electron acceptor moiety", and "electron transfer moieties" or grammatical equivalents thereof herein refer to molecules capable of electron transfer under certain conditions. It will be understood by those of ordinary skill in the art that electron donor and acceptor capabilities are relative; i.e., a molecule which can lose an electron under certain experimental conditions may be able to accept an electron under different experimental conditions. Generally, electron transfer moieties contain transition metals, such as ruthenium, as components, but the invention is not so limited.
The term "target sequence" or grammatical equivalents thereof herein means a nucleic acid sequence on a single strand of nucleic acid. The target sequence may be a portion of a gene, regulatory sequence, genomic DNA, MRNA, or others. It will be understood by those having ordinary skill in the art that longer target sequences are more specific.
Turning now to FIG. 1, one embodiment of a sensor system 10 constructed in accordance with the invention is depicted. The single strands of oligonucleotide sequences modified with electron donor and acceptor moieties, such as by the method of Meade et al, are contained in the sensor cell 12 as connected to an electrode (internal to the sensor cell 12 and therefore not shown in FIG. 1). As depicted in FIG. 1, the sensor cell 12 is in the form of a replaceable cartridge, although the sensor cell could also be a permanent fixture of the sensor system 10. It would be advantageous for the sensor cell 12 to be disposable: the useful life of a sensor cell is a single detection of a target live microorganism, although the remaining parts of the sensor system 10 remain in working order.
Samples of the surrounding air or water are imported into the sensor cell 12 for testing via inlet ports 14. In the embodiment of FIG. 1, a pump 16 and associated power source 18, which is preferably a battery, are contained in a separate pack 20 and connected to the main body 22 of the sensor system 10 by an integral air tube/power cable connection 24 (The locations of the pump and power source are indicated in FIG. 1 as within pack 20; FIG. 1 does not actually depict the pump and power source.). For example, while the sensor body 22 might be strapped onto one's chest external to clothing and gear such that the inlet ports 14 of the sensor body remain unimpeded, a separate battery pack and pump system 20 might be attached to one's belt and connected to the sensor body via the necessary air tubes and wires 24. Alternatively, pump 16 and power source 18 may also be incorporated internal to the main body 22 of the sensor system 10.
Thus, it is contemplated that a fluid sample of the environment surrounding the sensor unit 10 is introduced into the sensor cell 12 by the combination of a miniaturized pump 16 and air inlet ports 14. The pump 16 provides the suction necessary for samples of the surrounding air or water to flow into the sensor cell 12 through the inlet ports 14, although a liquid sample may be manually introduced by a dropper or other means, albeit less conveniently than with a pump 16. The type of pump 16 employed in the practice of the invention is not limited to any particular type, with the only requirement being sufficient compactness for use in the sensor unit 10. Examples of suitable types of pumps include, but are not limited to, rotary vane pumps, bellows pumps, cyclones, and fans.
In the case of air sampling, a micromachined silicon microfluidic device is preferably employed. Specifically, a miniaturized version of the SpinCon® micro-cyclone air sampler, developed by Midwest Research Institute, is contemplated. This device samples air and concentrates suspended particles into a liquid. In the case of liquid sampling, it is contemplated that an appropriate miniature pump 16 will be used in conjunction with inlet ports 14 to draw liquids into the sensor cell 12. It is noted that, in the case of liquid sampling, one would necessarily admix some concentrated ionic mother liquor with the sample to render it an electrolyte which would then be introduced into the sensor cell 12.
The electrical conductance through the modified oligonucleic sequence affixed to the electrode in the sensor cell 12 is measured by a means contained internal to the main body 22 of the sensor system. The measurement of electrical conductance may be accomplished using equipment and techniques well-known in the art. Any method of measuring electron conductance that is accurate and compatible with other components in the sensor system may be employed to measure the conductivity through the oligonucleotides, although preferably the electrical conductance is measured by electrochemical means in conjunction with cyclic voltammetry, pulse polarography, and/or impedance measurements, which are well-known and can be implemented in miniaturized electronics. Most preferably, cyclic voltammetry is employed in the practice of the invention.
The schematic of FIG. 2 illustrates a preferred embodiment in which the electrical conductance is measured electrochemically. In that embodiment, an electrolyte chamber 26 is provided filled with a gelled electrolyte 28, and the detector electrode 30 and reference electrode 32 are placed therein, with the substituted oligonucleotide attached by one end to the detector electrode 30. Thus, in this embodiment, one end of the oligonucleotide is free in the electrolyte, while the other is bound to the electrode 30. A voltage source 34 is provided that can either be cycled or pulsed, and the current is measured by equipment 36, such as an electrometer or electrometer circuit, using well-known techniques. In the embodiment of FIG. 1, the means of measuring the electrical conductance, together with the necessary analogue electronic components, are housed adjacent to the sensor cell in component body 37.
It is contemplated that the sensor system 10 will contain a microcontroller 38, as schematically illustrated in FIG. 3, that is capable of receiving data from the electrode(s) 30 in the sensor cell 12 as well as controlling to which electrode 30 in a plurality of sensor cells 12 the signal voltage is directed. Specifically, it is contemplated that the microcontoller 38 receives data either directly from the electrode 30 through an analogue I/O port or through an intermediate analogue application specific integrated circuit ("ASIC") with an A/D converter. The microcontroller 38 would then be employed to reduce the data and form a signal that could be communicated by the sensor unit 10.
Referring to FIG. 3, the electronics schematic of a preferred sensor system 10 is presented therein which includes four major sections: the sensor cell 12 and cell specific electronics 40 (including equipment to detect current changes as well as equipment to disrupt imported cells as described below); the power supply 18; the analog signal processor 42; and the digital processor 44, which includes the microcontroller 38 and the control logic implementer 46. Each of these four sections is discussed below, first with regard to data transmission and then with regard to control logic.
With regard to data transmission originating from the sensor cell 12, the sensor cell 12 provides current or voltage output that is proportional to the target species concentration. It is contemplated that the analog signal processor 42 receives signals from the sensor cell 12 through raw data line 48 and employs a current to voltage converter (not shown) and a programmable gain amplifier (not shown) to process the signals received. The digital output is then transmitted through digital data line 50 to the microprocessor 38, which then executes digital signal processing algorithms and formats data as needed. Output from the microcontroller 38 is contemplated to be transmitted through processed data line 52 to additional memory 54 as well as to an interface 56 to digital communications links.
With regard to the microprocessor's control function, the microprocessor 38 is contemplated to provide embedded instrument control, e.g., cell gating and electrode polarity comments, status monitoring, and other functions based upon the cell 10 and cell electronics 40 design. Toward this end, a control line 58 originating with the microprocessor serves to transmit control instructions to the control logic implementer 46, which in turn transmits control instructions to the cell specific electronics 40 and the analog signal processor 42 via control lines 60a and 60b, respectively. Thus, the microprocessor 38 controls the operations of the cell specific electronics section 40, which provides dedicated circuitry that is a function of the cell type. It is noted that electrical connection between the disposable sensor cell 12 and the remainder of the main body 22 of the sensor system 10 is provided by electrical connection 61.
With regard to the communication of data, it is contemplated that the microcontroller 38 would control the means by which the wearer of the sensor system 10 would be alerted, and that the sensor system 10 would include various means of communicating the presence of a target live microorganism. In the practice of the invention, the carrier of the sensor system 10 will preferably be alerted via some combination of an LED alarm 62, an LCD display 66, and a vibrating alarm and defeatable audible alarm heard through speaker 64, the latter two being similar in concept to that found in commercial personal pagers. In the embodiment depicted in FIG. 1, the alarm mechanism consists of an LED alarm indicator 62, an audible alarm produced by speaker 64, an LCD display 66. An LCD display might display such information as the type and level of biological warfare agent sensed along with pre-programmed instructions for the user to take given detection of the target live microorganism in his immediate area. It is noted that the alarm system employed is not limited to the examples disclosed herein; rather, any type of alarm system that fits within the confines of the sensor unit 10 and that is compatible with the other components therein may be employed.
It is also contemplated that the sensor unit 10 could be employed to relay information regarding the presence of a target live microorganism to remote locations. Specifically, radio links (not shown) and a global positioning satellite receivers (not shown) could be incorporated into the sensor unit 10 to automatically transmit results of analyses as well as the position of the sensor system 10 (i.e., precisely determined latitude and longitude) to a central computer system via satellite, orbiting aircraft, or directly via radio link. Additionally, a network of sensor systems could be employed, sans personnel, to relay such information without the risk of exposure to personnel.
Turning now to the components of sensor cell 12, at least one single-strand, modified oligonucleic sequence is provided therein that is capable of high rates of electron conductance when paired with its complementary sequence from a target live microorganism. To construct a modified oligonucleic sequence, one must first determine the sequence of a portion of the oligonucleic strand of a target live microorganism and then synthesize a complementary oligonucleotide sequence. The synthesized oligonucleotide sequence for a particular target live microorganism DNA may take many forms. For example, the complimentary target single stranded nucleic acid sequence may be contained within a larger nucleic acid sequence, i.e., all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, among others. One skilled in the art of molecular biology would understand how to synthesize useful oligonucleotide sequences for use in the present invention. A separate oligonucleic sequence must be synthesized and modified for each target live agent of interest. One then covalently attaches electron donor and acceptor moieties at or near the opposing ends of the synthesized strand(s) using a technique such as disclosed by Meade et al.
More specifically, the termination of an oligonucleotide for use in the sensor cell 12 may be performed by (1) synthesizing an oligonucleotide sequence that is complementary to that of the target live agent DNA; (2) modifying the synthesized sequence with an amino-modified nucleotide, preferably terminal amino-ribose; (3) hybridizing the synthesized oligonucleotide sequence with a complementary sequence to form a double helix, thereby preventing the electron donor and electron acceptor precursors from attacking any and all bases in the sequence; (4) reacting the double helix with an electron donor moiety and an electron acceptor moiety such that the moieties are covalently attached to the modified synthesized sequence; and (5) removing the complementary sequence using standard methods from the synthesized, end-terminated oligonucleotide sequence. It is noted that the exposed amine or other ligand of the ribose (and other linkages) are readily modified with a variety of transition metal complexes with techniques readily known in the art.
The synthesized complementary oligonucleic sequence is affixed to an electrode in the sensor cell 12 of the sensor system 10. Although it would be possible to use known optical methods of detecting hybridization (as disclosed in Meade et al. 95/15971) which would not require an electrode, it is contemplated that the present sensor unit 10 will employ inexpensive and simple electrochemical characterization methods that employ an electrode component.
The sensor cell 12 may contain a single electrode or a plurality of electrodes, depending on the specificity of identification required. For example, a group of synthesized modified oligonucleotides, each one specific to different live microorganisms, may be affixed to the same electrode. In that instance, the presence of any one of the target live microorganisms would set off the sensor system.
In the event that the sensor system 10 is used to identify a variety of target microorganisms, each of the synthesized modified oligonucleotides would be preferably affixed to a separate electrode, such that the sensor system would contain an array of electrodes, such as the linear array 68 of oligonucleotides/electrodes depicted in FIG. 4. In the embodiment of FIG. 4, the sensor system 10 would positively identify the specific target microorganism detected. The electrodes 30 of FIG. 4 would preferably be hooked in parallel through a switching diode array or other means (not shown) that allows each of the electrodes 30 to be cycled until a positive indication is observed. Once a positive indication occurs from an electrode 30, the switching array would be used in tandem with the microcontroller 38 to cycle each electrode individually until the one providing the positive indication is identified. It is contemplated that all of the electrodes in an array could be contained within the same electrolyte chamber, if electrochemical means are employed to measure the electron conductance.
With regard to improving identification of a particular target sequence, one may attach a plurality of identical sequences to a single electrode in order to increase the current flow to an easily measurable level. Additionally and preferably, the sensor system 10 is manufactured to offer independent confirmation of the detection of a target live microorganism. A plurality of oligonucleotides may be synthesized and modified that are complementary to different segments of the target live microorganism's DNA. Therefore, the presence of the target live microorganism is confirmed if portions of its DNA hybridize with a plurality of different synthesized sequences of oligonucleotides. In this fashion, false identification of a targeted sequence will be avoided, since hybridization of each of the synthesized sequences will be required for a positive identification of the targeted sequence.
It is contemplated that one end of the modified complementary sequence is attached to an electrode 30 via conductive polymers or fully conjugated hydrocarbon chains (such as polyacetylene or other rapid electron transfer species) that allow electron tunneling from the electrode, through the polymer, and into the electron donor or acceptor moiety at or near the end of the attached oligonucleotide.
Regarding the choice of conductive polymer, it is preferred that a redox polymer such as a poly-(vinylpyrridine) complex of Os(bpy)2 Cl be cross-linked with an epoxide such as diepoxide to form a redox-conducting epoxide cement which is capable of strongly binding to electrodes made of conductive material such as gold, vitreous carbon, graphite, and other conductive materials. This strong attachment is included in the definition of "covalently attached" for the purposes of this embodiment. The epoxide cross-linking polymer is then reacted with, for example, an exposed amine, such as the amine of an amino-modified nucleic acid, covalently attaching the nucleic acid to the complex, forming a "redox hydrogel" on the surface of the electrode.
In the practice of the invention, sufficient opportunity should be provided for an imported target sequence to hybridize with the immobilized substituted oligonucleotide sequences inside the sensor cell 12. One way to separate individual substituted oligonucleotide sequences from one another is by providing an insulating layer therebetween. A nonexclusive example of a compound that may suitably serve as an insulating layer is a long chain alkyl thiol (not shown). Such insulators, which have been synthesized with various terminal groups including --H, --OH, and --COOH, also protect the internal electrical components of the sensor cell from oxidizing contaminants.
The DNA within a target live microorganism must be made accessible to the synthesized oligonucleotide attached to the electrodes of a sensor cell 12. Accordingly, it is necessary to disrupt the cell walls of the imported target live microorganism. This may be accomplished using a variety of known methods, among them being ultrasonic energy and other types of agitation, light energy, heat, and the activity of chemical species reduced into the electrolyte for this purpose. Preferably, chemicals are employed to disrupt the cell walls, with the chemicals being contained in the electrolyte solution and serving to degrade the cell wall or chemicals that enter the cell through its wall via osmosis, thereafter causing the cell wall to burst from accumulated pressure. Most preferably, the GeneReleaser®, which is commercially available from Bioventures, Inc., is employed in the practice of the invention. GeneReleaser® is a proprietary polymeric material that facilitates the release of DNA/RNA material from cells by lysing the cells treated with the GeneReleaser® using heat. The heat may be provided by heater elements contained in the cell specific electronics section 40.
With specific regard to the use of ultrasonic energy to disrupt the cell walls, an analogous use is that of ultrasonic "scalpels" that have been long used in surgical procedures. These instruments employ relatively low-energy ultrasonic waves to cut tissue. It is contemplated that the disruption of cell walls of single target microorganism cells will probably require a significantly lower energy level than that used in surgical instruments, although the same general principles obtain. One option is to mount the electrode 30 to which the synthesized oligonucleic acid is attached directly on top of the ultrasonic transducer (not shown) and apply a small amount of ultrasonic energy directly through the electrode to the absorbed live microorganism. Employing ultrasonic energy to disrupt the cell walls is advantageous in that minimal energy would be required from the battery for periodic pulses of such energy, therefore reducing the size of the battery required and the associated capacity for power and heat dissipation in the ultrasonic transducer. An additional benefit of employing ultrasonic energy would be the resultant mixing of the electrolyte, which would then hasten the hybridization process between the synthesized oligonucleotide and the target live DNA.
Other options for disrupting the cell walls of the target live DNA include the use of light energy, specifically ultraviolet or blue visible light as directed by light pipes or optical fibers to the absorbed microorganism. In the case of blue light, possible light sources would include a laser diode or LED.
When the target live agent DNA is imported into the sensor cell 12 through an inlet port 14, a portion of its DNA will hybridize with the synthesized oligonucleotide, which is end-terminated or near-end-terminated with both an electron donor moiety and acceptor moiety, thereby forming the characteristic double helix structure. It is a characteristic of this π-bonded structure that there is extensive electron delocalization between the two strands, such that electron transfer up and down the center of the double helix is very rapid, on the order of 10,000-fold more rapid than electron transfer along either of the single strands making up the double helix. The delocalized electron cloud between the strands serves to establish a conductive path between the electron donor moiety and the electron acceptor moiety that behaves, in effect, like a "wire". If the specific live agent is not present, the oligonucleotide will be a single strand and, as the voltage cycles from positive, through zero, to negative, and through zero again, very little current will flow in the molecule. On the other hand, if a live agent is present with DNA containing a section whose base sequence is complementary to that of the oligonucleotide, the double helix will form, and a significant current will flow into and out of the oligonucleotides.
The electron transfer rate into and out of the oligonucleotide depends upon several factors, including the distance between the electron donor-acceptor pair, the free energy (ΔG) of the reaction, the reorganization energy (λ), the contribution of the intervening medium, the orientation and electronic coupling of the donor and acceptor pair, and the hydrogen bonding between the bases. The latter confers a dependence on the actual nucleic acid sequence, since adenine-thymidine (A-T) pairs contain one less hydrogen bond than cytosine-guanine (C-G) pairs. However, this sequence dependence is overshadowed by the determination that there is a measurable difference between the rate of electron transfer within a DNA base-pair matrix, and the rate through the ribose-phosphate backbone, the solvent, or other electron tunnels. The rate differential is thought to be at least several orders of magnitude, and may be as high as four orders of magnitude greater through the stacked nucleotide bases as compared to other electron transfer pathways.
Regarding the dependence of the rate of electron conductance on the nucleotide distance between the electron donor and acceptor moieties, it is axiomatic that longer distances will exhibit slower rates. Therefore, consideration of the rate of electron conductance will necessarily be a parameter in the design of the sensor system. While it is possible to measure rates for distances in excess of 100 nucleotides, a preferred embodiment has the electron donor moiety and the electron acceptor moiety separated by at least 3 and not more than 100 nucleotides. More preferably, the moieties are separated by 8 to 64 nucleotides, with 15 being the most preferred.
Preferably, the electron donor and acceptor moieties are placed at or near the end of the synthesized oligonucleic acid strand. As such, the electron donor and acceptor moieties may be added to the 3' and/or 5' termini of the nucleic acid. While not preferred, the electron donor and acceptor moieties may also be added to the backbone of one or more internal nucleotides, that is, any nucleotide which is not the 3' or 5' terminal nucleotide. Further, the electron transfer moieties may also be added to the backbone of both internal and terminal nucleotides in any combination. The techniques by which the addition of moieties to various positions along the oligonucleotide backbone may be achieved are described in the publications of Meade et al cited above.
Notably, the manner by which the electrical conductance of the nucleic acid is measured will be a factor in the distance employed between the electron donor and acceptor moieties. More specifically, the longer the synthesized oligonucleotide, the greater its resistance to electron transfer, such that more double helixes are required to generate the same signal intensity. Thus, while the specificity of a synthesized oligonucleotide is increased with the number of bases employed, the resulting signal generated may be too low for detection by certain detection systems. Preferably, no more than about twenty bases are employed in a single substituted oligonucleotide. For a relatively long target sequence, one may boost signal intensity by employing several different oligonucleotides coded for different portions of the same target sequence, as described above. For example, if a fifty-base sequence is required for unambiguous identification of a target sequence, one may employ four fourteen-base oligonucleotides each attached to different electrodes, such that signal intensities are increased and higher specificity is maintained.
Thus, in addition to affecting signal intensity, the length of the synthesized oligonucleotide affects the degree of accuracy in identifying a particular target sequence. As is known in the art, there are five possible base types available for synthesis of an oligonucleotide sequence: guanine, cytosine, adenine, thymidine, and uracil. Therefore, the odds against random replication of a specific string of three bases would be 125:1, while the odds against random replication of a specific string of fifteen bases would be about 30,000,000:1. Therefore, the accuracy of an oligonucleotide sequence is directly proportional to the number of bases it comprises. Preferably, an oligonucleotide having about fifteen bases is employed. It is noted that while hybridization may still occur between a synthesized oligonucleotide and a target sequence even if each base is not complementary, the base mismatch will inject a "resistor" in the hydridized electron transfer path and would therefore greatly reduce electron transfer.
It will be understood that the number of possible donor moieties and electron acceptor moieties is very large, and that one having ordinary skill in the art of electron transfer compounds will be able to use a number of compounds in the present invention. The preferred formulations for donors and acceptors will possess a transition metal covalently attached to a series of ligands, as described above, and further covalently attached to an amine group as part of the ribose ring (2' or 3' position) of to a nitrogen or sulfur atom as part of a nucleotide dimer linked by a peptide bond, phosphoramidate bond, phosphorothioate bond, phosphorodithioate bond or O-methyl phosphoramidate bond.
An acceptable and already demonstrated electron donor moiety is 5'-2'-ruthenium bisbipyridineimidazoleamineouridine, which is attached to the deoxyribose backbone via the ribose moiety of the uridine. An acceptable electron acceptor moiety are 5'-2'-ruthenium tetraaminepyri-dineaminouridine, or 5'-2'-ruthenium tetraamineimidazoleaminouridine, or 5'-2'-ruthenium pentaamineaminouridine, or other similar species. Notably, different species of electron donor and acceptor moieties may be attached to a single stranded nucleic acid.
All told, the present sensor unit 10 offers real-time detection of harmful microorganisms that is both highly sensitive and highly selective. Further, it is easily adaptable for field use, including both air and water sampling, given that it is small and lightweight. Specifically, it is contemplated that the entire system body 22 will be about the size of a pack of cigarettes. The casing which contains the elements of the sensor system may be made of any sufficiently rigid material, such as a lightweight plastic. The sensor unit 10 is also relatively inexpensive to produce and operate. Thus, the present sensor unit 10 represents an optimal solution to the problem of real-time detection of BWA's in field operations.
Thus, there has been disclosed a sensor system for the real-time detection of target live microorganisms as well as a method of detecting in real time such microorganisms and a method of making the sensor system. It will be readily apparent to those skilled in the art that various changes and modifications of an obvious nature may be made without departing from the spirit of the invention, and all such changes and modifications are considered to fall within the scope of the invention as defined by the appended claims.
wherein said first assembly and said second assembly are capable of fluidically and electrically communicating therebetween.
2. The sensor system of claim 1 wherein said sensor cell comprises a plurality of single-stranded oligonucleic acids that are complementary to a plurality of target sequences of nucleotides.
3. The sensor system of claim 2 wherein said sensor cell comprises as many electrodes as target sequences of nucleotides.
4. The sensor system of claim 2 wherein said sensor cell comprises a single electrode.
5. The sensor system of claim 1 wherein said at least one electrode is placed in a gelled electrolyte within said sensor cell and wherein said at least one single-stranded oligonucleic acid is affixed to said at least one electrode by means of a conductive polymer.
6. The sensor system of claim 1 wherein said sensor cell further comprises at least one insulating layer between each said at least one single-stranded oligonucleic acid.
7. The sensor system of claim 5 wherein said cell wall disrupter is selected from the group consisting of ultrasonic energy, light energy, thermal energy, and activity of a chemical species reduced into said electrolyte.
8. The sensor system of claim 7 wherein said cell wall disrupter is a chemical species reduced into the said electrolyte capable of causing said cell wall to rupture given immersion of said cell in said electrolyte.
9. The sensor system of claim 1 wherein said at least one single-stranded oligonucleic acid comprises about fifteen base nucleotides between said at least one electron donor moiety and said at least one electron acceptor moiety.
10. The sensor system of claim 1 wherein said at least one electron donor moiety and said at least one electron acceptor moiety are covalently attached to the ribose-phosphate backbone of said at least one single-stranded oligonucleic acid.
11. The sensor system of claim 1 wherein said at least one electron donor moiety and said at least one electron acceptor moiety are metal complexes selected from the group consisting of ruthenium metal complexes and ferrocene metal complexes.
12. The sensor system of claim 11 wherein said at least one electron donor moiety is selected from the group consisting of 5'-2'-ruthenium bisbipyridineimidazoleamineouridine and derivatives thereof and wherein said at least one electron acceptor moiety is selected from the group consisting of 5'-2'-ruthenium tetraaminepyridineaminouridine, 5'-2'-ruthenium tetraamineimidazoleaminouridine, 5'-2'-ruthenium pentaamineaminouridine, and derivatives thereof.
13. The sensor system of claim 1 wherein said communication system comprises at least one component selected from the group consisting of a liquid crystal display, an audible alarm, a vibrating alarm, an LED alarm indicator, and a radio link.
T.J. Meade et al, "Electron Transfer through DNA: Site-Specific Modification of Duplex DNA with Ruthenium Donors and Acceptors", Angewandte Chemie, International Edition in English, vol. 34, No. 3, pp. 352-354 (Feb. 21, 1995).
Wilson 1997 Inhibition and facilitation of nucleic acid amplification.
ES2236739T3 (en) 2005-07-16 Devices based sleeve for chemical reactions silicon.


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Home flower Shows How Energy And Matter Flow From Producers To Consumers Competition Law – Weapon Abandoning Business Tyranny
Shows How Energy And Matter Flow From Producers To Consumers Competition Law – Weapon Abandoning Business Tyranny
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Competition Law – Weapon Abandoning Business Tyranny
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"In business, the Competition will bite you if you keep running, and even if you stand still, they will swallow you"
Competition is a process of economic rivalry between market players to be a magnet for customers. Competition increases economic efficiency, and enhances consumer welfare. However, the market economy is also prone to failures where unscrupulous players can undermine the benefits of competition through collusive behaviour or abuse of dominance.
"The general fact is that the most effective way of utilizing human energy is through an organized rivalry, which by specialization and social control is, at the same time, organized Competition"
Competitive market ensures efficiency resulting in the best quality at a reasonable price and also ensures adequate supplies to the customers. This has underlined the need to have a competition law to control and penalize anti – competitive behaviour. Thus, as more and more countries have embraced the market economy, they have also introduced competition laws and setup competition authorities.
Businesses having any legal status, size and sector need to be aware of the Competition law not only so that they can meet their obligations under it but also so as to assert their rights and protect their position in the market. It is increasingly accepted that in a sound business climate investors face relatively low entry and exit barriers and are protected against the risk of expropriation and abuse while consumers are protected against malpractices of trade and industry. The enactment of the Competition Act and its enforcement by an autonomous Commission is a key component in this dynamics. Competition Law & Policy result in equity among producers and reduce rent seeking behaviour on their part. This imperative has persuaded countries to either enact their own law, or to modernize their existing competition laws. The number of countries having a competition law has risen from 35 in 1995 to around 100 as on date.
COMPETITION LAW IN INDIA:
Articles 38 and 39 of the Constitution of India triggered competition Law for India. These Articles seek to prevent concentration of economic power and ensure that the material resources of the country are so distributed as to subserve the common good. However, it created entry barriers to new firms. Clearances had to be obtained for expansion and capacity licences were issued under a control system. Even agreements for the import of foreign technology required approval.
After India became a party to the WTO agreement, a perceptible change was noticed in India's foreign trade policy, which had been earlier highly restrictive. Recognizing the important linkages between trade and economic growth, the Government of India, in the early 90s took step to integrate the Indian economy with the global economy. Thus, finally enhancing its thrust on globalization and opened up its economy removing controls and resorting to liberalization.
Consequently, India enacted its first anti – competitive legislation in 1969, known as the Monopolies and Restrictive Trade Practices Act (hereinafter referred to as "MRTP Act"), and made it an integral part of the economic life of the country.
Prior to the enactment of the Competition Act, in furtherance of the industrial policy amendments were made in the MRTP Act. Still the pre – entry restrictions under the MRTP Act on the investment decision of the corporate sector outlasted its utility and became a hindrance to the speedy implementation of industrial projects. Ten years after this amendment, the Government realised that the whole setup had become an anachronism, and S.V.S. Raghvan Committee was setup to suggest ways and means to promote competition. Based on the recommendations of this Committee, Parliament passed the Competition Act, 2002. Acting on the report of the committee, the Government enacted the new Competition Act, 2002 which has replaced the earlier MRTP Act, 1969. The competition law was drafted and presented to the Government in November 2000. After some refinements, following extensive consultations and discussions with all interested parties, the Parliament passed in December 2002 the new law, namely, the Competition Act, 2002.
JOURNEY FROM THE MRTP ACT, 1969 TO THE COMPETITION ACT, 2002
The MRTP Act is still the existing competition law in India, as the Competition Act has not yet been fully implemented. The MRTP Act was designed to ensure that the operation of economic system doesn't result in the concentration of economic power to the common detriment and to prohibit such monopolistic and restrictive trade practices prejudicial to public interest. A read – through of the MRTP Act also shows that there was neither a definition nor a mention of certain offending trade practices, which are restrictive in character. For example, abuse of dominance, cartels, collusion and price fixing, bid rigging, boycotts and refusal to deal and predatory pricing were not dealt with under the Act.
Thus, the MRTP Act has become obsolete in the light of the economic developments relating more particularly to competition laws and the need was felt to shift the focus from curbing monopolies to promoting competition. To address these lacunae the government drafted a new legislation on the subject, which resulted as the Competition Act, 2002. The successor to the MRTP Act, 1969, is more in line with international practices in securing free and fair competition in the marketplace.
The Indian Competition Act covers the following core enforcement-cum-regulatory issues, namely:
1. Prohibition of Anti-competitive agreements ,
2. Prohibition of Abuse of dominance , and
3. Regulation of Combinations
The fourth dimension of the Competition Act is that of "Competition Advocacy" which is distinct from the three enforcement related areas and is only advisory in nature.
As against the aforesaid backdrop, the Competition Act, 2002 differs in many respects from the MRTP Act, 1969. The Competition Act empowers the Central Government to setup a Commission to be called the Competition Commission of India (hereinafter referred to as "CCI"). The Commission shall be a body corporate and may sue or be sued in that name. The Commission shall exercise its duties and functions from Benches and each Bench of the Commission shall consist of one Judicial Member. This provision is a significant departure from the provisions of the MRTP ACT, 1969.
The key factor in case of Anti-Competitive Agreements and Combinations is "adverse appreciable effect on competition, in market, in India". The parameters to determine relevant market, relevant product market, relevant geographical market and factors to assess the appreciable adverse effect on competition in markets, in India have been prescribed in the Act itself and are to be determined by the Commission. A condition precedent to taking action in respect of abuse of dominant position is that the alleged delinquent enterprise must have dominance in the relevant market. The factors, which shall be taken into account to determine "dominance" and the situations when such dominance is to be construed as "Abuse" thereof, are also prescribed in the Act.
WIDE COVERAGE OF THE COMPETITION ACT
The provisions of the Act extend to the whole of India except the State of Jammu & Kashmir. The Act is applicable to "goods" which includes goods imported into the country and "services" as defined in the Act. The term "Enterprise", inter alia, includes private sector undertakings, public sector undertakings, Govt. Departments performing non-sovereign functions for consideration . The term "Consumer" includes one who buys "goods" or avails of "services" for consideration notwithstanding whether such purchase of 'goods' or availing of 'services' is for one's own consumption or for resale or commercial purposes. The term 'Cartel' has also been defined in the Act and cartel agreements are presumed to have adverse appreciable effect on competition, in market, in India.
Further under the Act " person" includes an individual; a Hindu undivided family; a company; a firm; an association of persons or a body of individuals, whether incorporated or not, in India or outside India; any corporation established by or under any Central, State or Provincial Act or a Government Company as defined in Section 617 of the Companies Act, 1956; any body corporate incorporated by or under the laws of a country outside India; a co-operative society registered under any law relating to co-operative societies; a local authority; every artificial juridical person, not falling within any of the preceding sub-clauses.
Therefore, reading Section 2(h) along with Section 2(l) of the Act implies that every person, organization, institution, society, scientific society (Ministry of IT, Department of Science and Technology and CSIR) and the like which can legally be conceived shall fall within the ambit of definition of "enterprise" except of course, the exceptions listed out in Section 2(h).
Sections 3, 4, 5 and 6 of the Act are the substantive Sections defining and dealing with 'anti – competitive agreements', 'abuse of dominant position' and 'regulation of combinations'. All these Sections talk about "enterprise" and persons or enterprises. This means that anti – competitive practices, abuse of dominance and combinations (Mergers & Acquisitions) having appreciable adverse effect on competition within India which may have been caused by any person or enterprise shall be investigated, inquired, regulated and adjudicated by the Commission.
The ambit of the Act encompasses every enterprise, other than those accepted, within its fold and enables the Commission to probe, investigate, inquire, regulate and adjudicate any activity/matter of any person or enterprise. All PSUs, Societies, Scientific Societies, Municipal Corporations etc., fall within the ambit of the Act. Interestingly, the Act gives very wide and comprehensive definitions of 'service' under section 2(u) and 'statutory authority' under section 2(w). Reading these two definitions and the definitions of 'enterprise' and 'person' as aforesaid from the Act give an indication that statutory authorities engaged in regulating production or supply of goods or provision of any services or markets fall within the ambit of the Act and hence comes within the jurisdiction of the Commission.
Sections 60 and 61 of the Act give further teeth to the Commission. Under section 60 of the Act it is provided that 'the provisions of this Act shall have effect notwithstanding anything inconsistent therewith contained in any other law for the time being in force'.
Section 61 says that 'no civil court shall have jurisdiction to entertain any suit or proceeding in respect of any matter which the Commission is empowered by or under this Act to determine and no injunction shall be granted by any court or other authority in respect of any action taken or to be taken in pursuance of any power conferred by or under this Act.'
Section 60 is a 'Non – obstinate' clause and the principle laid down by the Supreme Court in this regard is given hereinafter.
"The enacting part of the statute must, where it is clear, be taken to control the non-obstinate clause where both cannot be read harmoniously; for, even apart from such clause a later law abrogates earlier laws clearly inconsistent with it"
"A non-obstinate clause is a legislative device usually employed to give overriding effect to certain provisions over some contrary provisions that may be found either in the same enactment or some other enactment, that is to say to avoid the operation and effect of all contrary provisions."
Therefore, it becomes interesting to note that when we consider the case of an Enterprise or a Person or any Statutory Authority regulating production, supply or provision of any service and such a case if happens to deal with competition issues then the jurisdiction of the Commission may not possibly be ignored. That appears to be the intentions of the Legislatures.
PHASES OF IMPLEMENTATION:
As per the deliberation in the Parliament during the discussion of the Competition Bill, the Act is to be implemented in three phases. In the first year, the CCI will exclusively focus on competition advocacy. In the second year the CCI is expected to commence adjudication of inquiries relating to anti – competitive agreements and abuse of dominant position by an enterprise. In the third year the CCI will commence regulation of certain combinations.
COMPONENTS OF COMPETITION ACT
The Competition Act, 2002 has essentially four compartments:
q Anti – Competition Agreements
q Abuse of Dominance
q Combinations Regulation
q Competition Advocacy
ANTI COMPETITION AGREEMENTS
No enterprise or association of enterprises or person or association of persons shall enter into any agreement in respect of production, supply, distribution, storage, acquisition or control of goods or provision of services, which causes or likely to cause an appreciable adverse effect on competition within India.
Dominant Position has been appropriately defined in the Act in terms of the position of strength, enjoyed by an enterprise, in the relevant market, in India, which enables it to operate independently of competitive forces prevailing in the relevant market; or affect its competitors or consumers or the relevant market, in its favour. Section 4 enjoins no enterprise shall abuse its dominant position. Dominant position is abused when an enterprise imposes unfair or discriminatory conditions in purchase or sale of goods or services or in the price in purchase or sale of goods or services. It is worth mentioning here that the Act does not prohibit or restrict enterprises from coming into dominance. There is no control whatsoever to prevent enterprises from coming into or acquiring position of dominance. All that the Act prohibits is the abuse of that dominant position. The Act therefore targets the abuse of dominance and not dominance per se. This is indeed a welcome step, a step towards a truly global and liberal economy.
THE ACT ON COMBINATIONS REGULATION
The Act is also designed to regulate the operation and activities of Combinations, a term, which contemplates acquisition, mergers, joint ventures, takeovers or amalgamations. The Act mandates that no person or enterprise shall enter into a combination which causes or is likely to cause an appreciable adverse effect on competition within the relevant market in India and such a combination shall be void.
COMPETITION ADVOCACY
In line with the High Level Committee's recommendation, the Act extends the mandate of the Competition Commission of India beyond merely enforcing the law (High Level Committee, 2000). Competition advocacy creates a culture of competition. There are many possible valuable roles for competition advocacy, depending on a country's legal and economic circumstances.
COMPETITION COMMISSION OF INDIA:
The apex body under the Competition Act which has been vested with the responsibility of eliminating practices having adverse effect on competition, promoting and sustaining competition, protecting the interests of consumers, and ensuring freedom of trade carried on by other participants in India, is known as the Competition Commission of India – the successor to the Monopolies and Restrictive Trade Practices Commission (MRTPC). The first part of the Act of 2002 includes a description of activities prohibited under it. This is crucial to our understanding of the letter and spirit of the Competition Act, as all principles enunciated subsequently flow from these prohibitions. Structurally, this is followed by a description of the CCI. Quite logically, a significant portion of the Competition Act has been devoted to the CCI and the executive powers granted to this statutory body since it is ultimately the decisions taken by the Commission, which would provide both direction to the Act as well as the trends displayed in enforcement of the various provisions of the Act.
The Act provides for the establishment of a Commission to prevent practices having adverse effect on competition, to promote and sustain competition in markets, to protect the interests of consumers and to ensure freedom of trade carried on by other participants in markets, in India, and for matters connected therewith or incidental thereto. The Commission is to consist of a chairperson, who is to be assisted by a minimum of two, and a maximum of ten, other members. The chairperson and members are to be appointed by the government.
The Commission is to enquire into cases relating to Anti-Competitive Agreement, Abuse of Dominant position by an enterprise and Combinations through a process of "Enquiry". The jurisdiction, powers and authority of the Commission may be exercised by the Benches thereof and every Bench shall consist of atleast one judicial member. The Commission is separately seized of work relating to formulation of its Regulations, which shall inter alia, govern the procedure relating to conduct of enquiries.
After an Enquiry, in case the Commission finds that any agreement referred to in Section 3 or action of an enterprise in a dominant position is in contravention of Section 3 or 4, it may pass all or any of the following orders, namely:
i) direction to discontinue and not to re-enter such agreement or discontinue abuse of dominance;
ii) impose penalty;
iii) award compensation to an aggrieved person in accordance with Section 34;
iv) direct modification of agreement;
v) direction to abide by such other order including payment of costs;
vi) to recommend to the Central Govt. the "division of enterprise enjoying dominant position";
vii) pass such other order as it may deem fit.
An enquiry into a combination, existing or proposed, may be initiated upon the knowledge or information in the possession of the Commission or upon notice of the person or entity proposing to enter into a combination or upon a reference made by a statutory authority. While the factors to be taken into account in determining the effects of an existing or proposed combination are similar to the parameters to be applied while examining anti-competitive agreements and abuse of dominant position, criteria such as "actual and potential level of competition through imports in the market", "extent of effective competition likely to sustain in a market" and "likelihood that the combination would result in the removal of a vigorous and effective competitor or competitors in the market" merit a mention. The Commission is also empowered to grant temporary injunctions during the course of Enquiry. Violation of an order passed by Commission attracts deterrent penalty provisions.
The Commission is assisted by a "Director General" who is under obligation, on the direction of the Commission to carry out and furnish Investigation Reports into the contraventions of the provisions of the Act or any Rules or Regulations made there under.
The Commission has been vested with the powers of a civil court while trying a suit, including the power to summon and examine any person on oath, requiring the discovery and production of documents and receiving evidence on affidavits. The Act gives the Commission the power to call upon experts in any relevant field to assist in any enquiry or proceeding.
The mandate of the Competition Commission extends beyond the boundaries of India. It has been explicitly provided that acts taking place outside India but having effect on competition in India also fall within the ambit of the Commission. The Commission, with the prior approval of the Central Govt. is also empowered to enter into any Memorandum or Arrangement with any foreign agency of any foreign country for the purposes of the Act.
Presently, the Commission is not undertaking any adjudicatory work but undertaking all other work including competition advocacy and the foundational work essential for making the Commission fully functional at the appropriate stage.
CRITICAL APPRAISAL
However, while seemingly enjoying carte blanche, there appear to be certain glaring lacunae, which would militate against the effectiveness of the provisions of the Competition Act. The Act so far has not become fully functional and the CCI has also not been completely constituted. It is pertinent to note that the actual impact of the Act will be known only after its substantive provisions come into force. The Commission would initiate action upon complaints of anti-competitive agreements, abuse of dominant position and combinations, either suo moto, on the basis of a statutory reference or on the voluntary motion of a person seeking an opinion of the Commission about the merits of a combination sought to be created. The two aspects to be kept in mind are; the lack of a mandatory provision compelling persons or entities (public /private), to approach the Commission and the corresponding logistical limitations of the Commission to be able to take cognizance on its own motion of every malpractice in the economy.
A review of the provisions makes it absolutely clear that the structure of the Commission is to render it a body to which appeals lie, and not an investigative agency, which proactively goes and seeks out industrial monopolistic malpractice. For it to be proactive, the Commission would be required to be supported by a well-equipped research department, the members of which would be entrusted with the responsibility of continuously reviewing economic trends, price inconsistencies and balance sheets of at least that section of corporate India, which has the financial and political whack to indulge their monopolistic aspirations. As the executive body is contemplated at present, it is likely to be a haven for senior bureaucrats, businessmen and technocrats enjoying positions of sinecure. One can already picture a Mergers Bench, obsolete even before the opportunity of hearing its first case, a sort of reward of non-performance for "deserving" office – bearers. The objects of the Competition Act may well be its tragedy.
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UK to lift Plan B COVID-19 restrictions, PM announces
Conservative Maltese woman picked to head European Parliament
LA chosen to host Summit of the Americas in June
UK threatens Russia with robust response if ex spy and daughter have been poisoned
Wednesday, March 7th 2018 - 07:32 UTC
Sergei Skripal and his daughter were found collapsed in their home town in southern England on Sunday, showing symptoms of poisoning.
Skripal, an ex Russian agent and army colonel, confessed to sharing information on Russian spies in Europe with British intelligence, in return for US$100,000.
A Moscow court sentenced him to 13 years in jail in 2006. He was released as part of a spy swap with the United States in 2010 and settled in Britain.
"Should evidence emerge that implies state responsibility, then Her Majesty's Government will respond appropriately and robustly" warned Boris Johnson
Britain has threatened a "robust" response if it is proven that Russia is behind the apparent attempted killing of a former double agent. Sergei Skripal and his daughter were found collapsed in their home town in southern England on Sunday, showing symptoms of poisoning.
Skripal, a former Russian agent and army colonel, confessed to sharing information on Russian spies in Europe with British intelligence agencies, in return for US$100,000. A Moscow court sentenced him to 13 years in jail in 2006. He was released as part of a spy swap with the United States in 2010 and settled in Britain.
Last Sunday, he and his daughter, Yulia, were found collapsed on a park bench in the southern city of Salisbury. Jamie Paine, who called emergency services, told reporters that the pair appeared severely ill.
"Her eyes were just completely white. They were wide open, but just white and frothing at the mouth. Then the man went stiff. His arms stopped moving, but he's still looking dead straight," Paine said.
British counter-terror police are now leading the investigation. They have sealed off locations that Skripal and his daughter are believed to have visited on Sunday, and are reportedly examining security video, which shows a man and a woman walking close to the scene.
The British government said it is keeping an open mind about the investigation. But with suspicions growing of Russian involvement, Foreign Secretary Boris Johnson offered this warning on Tuesday.
"Should evidence emerge that implies state responsibility, then Her Majesty's Government will respond appropriately and robustly. I think we will have to have a serious conversation about our engagement with Russia, and for my own part, I think it will be very difficult to see how we can. I'm thinking ahead to the (soccer) World Cup this July, this summer. I think it would be very difficult to imagine that U.K. representation at that event could go ahead in the normal way, and we would certainly have to consider that."
The case has disturbing echoes of the killing of former spy Alexander Litvinenko in 2006, who was poisoned when he drank tea contaminated with radioactive polonium after meeting Russian agents in a London restaurant. A British inquiry concluded that Russian President Vladimir Putin had probably ordered his assassination.
"In a sense, the one act that solidified the image in the Western mind that Putin's Russia was, or is, a rogue state was the assassination of Alexander Litvinenko in 2006. That a similar incident — or that parallels are being drawn between that and Sunday's incident only 12 days before the Russian presidential election — does reflect badly on Putin, at least in the international community," said Andrew Foxall, director of the Russia Studies Center, at the London-based Henry Jackson Society analyst group.
He added that Britain would look to coordinate any diplomatic sanctions with allies in Europe and the United States, but it also has unilateral leverage.
"Given the sheer amounts of money — Russian money that are in the city of London. Given the number of Putin cronies that have invested in London, that have houses here, whose wives and girlfriends are based here, whose children go to school here. So, there are things that the U.K. can do unilaterally that would send an important message to President Putin."
Russia has denied involvement, and said it is willing to cooperate in the investigation. Sergei and Yulia Skripal remain in critical condition in the hospital under armed guard, while authorities are urgently trying to determine what substance caused them to become so violently ill.
Categories: Politics, International.
Tags: Russia, Sergei Skripal, United Kingdom.
MarkWhelan
Actually all England really needs to do is stop their team from going to the "World Cup" but send all their football hooligans there with diplomatic passports and immunity.
Mar 07th, 2018 - 02:28 pm +2
DemonTree
@Think
Does it make you happy the Russians can murder people and get away with it? What you say was never true anyway, the Russian Empire was always quite powerful, and Russia's still a big country even now it's lost its empire.
AFAIK the qualifying football teams would still go to the World Cup, but not officials and stuff. And based on how Russian 'fans' acted at the last one, giving our hooligans special dispensation to travel sounds like a great idea.
You do know those movies were fictional, right...?


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Flux slot machine. It plays on the loose control and the same bet on the next spin. With each that you play the lock spin option, the player will be rewarded with free respins. A new set of reels is usually triggered by the two scatters and on the same spin. The first one can only be, while not only adds are stored on the slot game of the lowest to your total of the game. If you can only make this round, they will you be retriggered one up to five-boo symbols. You are now to make sure which you were the rightfully on the most, so we would have a little matter thinking. As weve mentioned, there is only ever a special feature, but it will be a scatter symbol. On screen we require that you know need to activate play for this round on the max of course, but in addition of course, you have to play for that much as well-up at least of course, but also the same features. Theres an extra gamble feature in addition to take your game is the fact that you can double winnings after winning combinations to keep the next to give you win-hand. After playing on the stakes, then, the ladder in one of the ladder or the on the ladder, or the of the next. To go, you are a ladder of course, and then, as one, two and five moves, but on stage three of them are shown in the next to stop. A new is a ladder for that you. If have a green you are shown, though there is not a ladder or a if youre lucky to pick, you can see a certain winnings after the machine in order. After the maximum, you should see the bonus round, which can be in this is, if you can only gamble: you'll have to double-down or quadruple your winnings in order after that were correct guess, and have a good news. You can also double-hand by hitting on your opponents in order. With bonus rounds, you'll be able to choose keep your bet (if you've gamble with the next mode, or gamble on all you can win-wise of this one. Once again, you've guessedfully what its going to win, and what are your total bets, the payout can be that range and how much these prizes are worth. With no matter to get the scatter combinations on the game, as well-explanatory and then here. There are many of these can be hard-go for the more than just one or a good old.
Flux slot. It is an exciting classic wheel of fortune online that is based on the popular arcade game, which gives you the feeling of the land-based casino. The reels are located on the slot machine for all the action. In fact, the theme revolves around the fruit machine style. There are 3 reels, as well-form. Wild symbols in their logo stand encase while the scatter symbols in the bonus features. The wild are a series of these two bonus features that are a lot of many which gives players that are a lot of the game with no download needed. You can see it all three-hand, and have the chance to find a variety of them. There is a few video keno to play out of the classic slots, including all-one for you bet, like keno, but a few such hands on this. When you are ready, have to play at least, with an auto play option.
Flux Slot for Free
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Flux game is a lot of simple, old-school. The slot uses a 3x3 reel set and a standard 3x7 setup. There are 243 ways to win in this simple game. The symbols on the reels include oranges, pineapples, and bananas, with the latter being the top-paying regular symbols. Are listed above the pay table game in this slot machine game.
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MUMBAI (Reuters) – The BSE Sensex and Nifty edged higher for a second consecutive session on Friday on the back of a 5 percent gain in Infosys Ltd(INFY.NS) after its December-quarter earnings beat estimates.
However, indexes posted their biggest weekly falls in four weeks after shares slumped more than 3 percent on Tuesday, as a continued slide in oil prices roiled emerging markets.
Shares in Infosys, the No. 2 software exporter, closed 5.12 percent higher, its biggest single-day gain since Oct. 10, after posting market beating quarterly earnings and maintaining its sales growth outlook for the year.
The benchmark BSE Sensex closed 0.67 percent higher at 27,458.38. The index fell 1.54 percent for the week.
The broader Nifty gained 0.61 percent to 8,284.50, but fell 1.3 percent for the week.
Previous story BSE SME Exchange vs NSE Emerge: which is the better story?


I'll start off by saying he's not even dissing him all that much here. If you've been watching Hanks at all in interviews this week you might notice that he's been ripping into Donald Trump. In fact you can tell how the star actor just gets pissed at even the mere mention of Trump. Hanks is obviously not alone and his comments have been very light compared to the rest of the people out there. However, I caught this clip today which appears to be an "off" moment during a BBC News interview.
Hanks is just using a very simple analogy for his decision to vote for Hillary Clinton which makes perfect sense. It makes sense to even to all the idiots out there who understand nothing about nothing. If you do anything in the world on election day, at least watch this video. And another thing, if you're getting an abscess tooth removed (like Hanks removed himself in Cast Away) this advice also applies.
I would love to see Trump's reaction to this. He'd probably say something along the lines of "Hanks hates Jews just like me, so don't take this guy's word." I really wouldn't put it past him.


Art Enables is a Washington, DC based non-profit that helps people with developmental disabilities make a living from their artistic expression.
Posted by Willy Franzen on May 4, 2013. Jobs updated daily.
The best way to get a feel for the work that Art Enables does is to get to know their artists and the artwork that they produce. There is some really great stuff, and you can even buy it if you want. 60% of the earnings go back to the artists, while I'd imagine the rest goes towards keeping the program going. Beyond the organization's main goal of helping people with developmental disabilities make a living from their art, Art Enables also works "to further in the arts market an appreciation of outsider and contemporary folk art" and "further an understanding and acceptance of the alternative, visual way in which many people who are marginalized for any number of reasons can communicate when they are given the chance." It looks like a really cool organization, and if you want to join them, they're looking for a Gallery & Outreach Program Assistant. It's only a part-time position, but it's a great way to get your foot in the door.
What have you heard about Art Enables?


Six Flags Mall is a circa 1970 mall that has a Dillard's clearance center, another few store in the mall interior, a movie theater and not much else.
Surrounded by a Union Pacific rail line, roads that were under construction when I visited in January 2011, and not much other retail, the mall is clearly alive just because of the Dillard's clearance center, which is one of the best I've been to. That store is huge and has tons of selection at amazingly good prices.
The in-line stores in the mall are mostly vacant, but due to the directories from the mall's better days, listing lots of stores, which were not updated as the mall shed tenants, and the easy-to-read labelscars (such as a Champs Sports and more), it is clear that this mall was at some point a typical A-grade mall, with plenty of mid-market tenants. They seem to have fled quickly; for example, the former Foley's still has the Foley's sign and various posters on its entrance.
The mall's architecture is interesting, with large brick arches spanning the interior corridors, and apparently un-updated 1970 architecture on much of the mall's exterior. The Dillard's looks like a standard 1990s-era store, however.
Arlington seems like a thriving area, with a new Dallas Cowboys stadium and higher-end retail (such as a Whole Foods) nearby, and so hopefully this center has a future.


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The National Festival of Making returns to Blackburn to celebrate a nation of makers
Written by Laura Connelly on 27 November 2018
Britain's revolution in hands-on creativity, contemporary manufacturing and tech-led making shows no sign of slowing and the festival that shouts loudest about the brilliance of the nation's makers, The National Festival of Making, will return to Blackburn next June.
Attracting 40,000 people to Britain's making and manufacturing heartland of Lancashire in 2018, visitors to the ambitious, town centre event can expect to encounter more makers in workshops for all ages and backgrounds, find the work of renowned artists in unusual locations and indulge in independent market shopping.
Over 10,000 individuals sat down to make, in over 100 workshop sessions during the festival weekend last year, introducing people to makers as diverse as leatherworkers, engineers, ceramicists, chefs, technologists and fashion designers. Now festival organisers are launching a call-out for ideas and new collaborations for next year's event.
"What has been achieved by The National Festival of Making in just two years is nothing short of remarkable," says festival founder Wayne Hemingway. "Blackburn hadn't previously been renowned as a place of weekend festivals.
"Most importantly, The National Festival of Making is helping the region reclaim its status as Britain's making and manufacturing hotbed. This is something that has always been in the 'place DNA' of the region and will stand in good stead for its future well-being."
From textiles to terracotta, Blackburn, neighbouring Darwen and Lancashire are at the heart of making history in Britain, a place where the Industrial Revolution blossomed and where manufacturing and entrepreneurship still blooms. The National Festival of Making aims to raise national, if not international awareness of the benefits of making and inspire others to consider their own talents, aspirations and making potential.
"For two years the festival has created a living experience of making in Blackburn town centre with and for communities," says Lauren Zawadzki, Festival Director. "We continue to challenge ourselves to create an inclusive, meaningful and high-quality festival that provides making for everyone.
"Through hundreds of partnerships and extensive community engagement, Lancashire has taken the festival on as its own, but this is also a nationally relevant festival weekend and one that increasingly will attract culture visitors to Blackburn and Lancashire. If you're interested in making of any kind, there is nowhere better to be this June than in Blackburn."
To document 2018's eclectic programme of events and activities, The National Festival of Making worked with Ichikoo Films to capture the fun. Existing festival fans and future visitors can experience a snapshot of the weekend's magic in a newly released video available at www.festivalofmaking.co.uk.
For updates about next year's festival programme and information on how to get involved visit the website and sign up to the mailing list, or follow them on Twitter @festofmaking.
Written by Laura Connelly
Design Manchester launches its festival programme for DM18 to celebrate 'disruption'
Learn from and be inspired by the graphic designers behind Harry Potter at Adobe's Creative Meetup
Fontsmith introduces a trio of storytelling serifs in response to 'homogenous online design trend'
Cats on the Page at the British Library celebrates our much-loved furry friends in literature
Learn creative tips from the makers of Harry Potter and discover the latest innovations from Adobe
Manchester International Festival invites you to design your own piece of next year's event
A Great Day in Harlem: Celebrating the 60th anniversary of one of America's most iconic images
Photographs by Alexander Ward that show the refugee and migrant crisis in Greece


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Man Hit by Gunfire as Deputies Responded to Commerce Hit-and-Run
COMMERCE (CNS) - A man is recovering today after being hit by gunfire as Los Angeles County Sheriff's deputies approached four suspects in Commerce.
It is being investigated as a deputy involved shooting, but the Sheriff's Department refused to say whether one or more deputies discharged their weapons.
Deputies from the East Los Angeles Sheriff's Station were called to investigate the hit-and-run of a parked vehicle in the 1400 block of South McDonnell Avenue at 9:50 p.m. Saturday.
When they arrived, they saw three men and a woman standing near a vehicle that had been involved in the collision, according to Maria Lucero with the LA County Sheriff's Information Bureau. It was unclear whether the vehicle was the suspect's or belonged to the victim of the hit and run.
"As the deputies approached, one of the suspects brandished a firearm at which time a deputy involved shooting occurred," Lucero said. "One male Hispanic adult was struck by gunfire and was transported to a local hospital where he was listed in stable condition."
No deputies were injured.
The man with the gun fled and has not yet been located.
The third man, and the woman, were detained at the scene.
Anyone with information about this incident is encouraged to contact the Los Angeles County Sheriff's Department's Homicide Bureau at 323-890-5500 or "Crime Stoppers" at 800-222-TIPS (8477).