|Número de publicación||US8084245 B2|
|Tipo de publicación||Concesión|
|Número de solicitud||US 12/950,335|
|Fecha de publicación||27 Dic 2011|
|Fecha de presentación||19 Nov 2010|
|Fecha de prioridad||22 Oct 1993|
|También publicado como||CA2174648A1, CA2174648C, DE69425673D1, DE69425673T2, DE69433635D1, DE69433635T2, EP0734397A1, EP0734397A4, EP0734397B1, EP1025902A2, EP1025902A3, EP1025902B1, US5472672, US5529756, US5814700, US5837858, US7858364, US20030069411, US20050281719, US20090054605, US20110124529, WO1995011262A1|
|Número de publicación||12950335, 950335, US 8084245 B2, US 8084245B2, US-B2-8084245, US8084245 B2, US8084245B2|
|Inventores||Thomas M. Brennan|
|Cesionario original||The Board Of Trustees Of The Leland Stanford Junior University|
|Exportar cita||BiBTeX, EndNote, RefMan|
|Citas de patentes (6), Clasificaciones (65), Eventos legales (4)|
|Enlaces externos: USPTO, Cesión de USPTO, Espacenet|
This is a continuation of U.S. application Ser. No. 12,177,768, filed on Jul. 22, 2008, which is a continuation of U.S. application Ser. No. 11/022,377, filed on Dec. 21, 2004, which is a continuation of U.S. application Ser. No. 10/171,497 filed on Jun. 12, 2002, which is a continuation of U.S. application Ser. No. 09/689,272, filed on Oct. 10, 2000, which is a continuation of U.S. application Ser. No. 09/162,348, filed Sep. 28, 1998, which is a continuation of U.S. application Ser. No. 08/452,967 filed May 30, 1995, now U.S. Pat. No. 5,837,858, issued Nov. 17, 1998, which is a continuation of U.S. application Ser. No. 08/453,972, filed May 30, 1995, now U.S. Pat. No. 5,814,700, issued Sep. 29, 1998, which is a divisional of U.S. application Ser. No. 08/142,593, filed on Oct. 22, 1993, now U.S. Pat. No. 5,472,672, issued Dec. 9, 1995, the contents all of which are incorporated herein by reference.
The present invention relates, generally, to polymer synthesis apparatus, and, more particularly, relates to polymer synthesis apparatus using arrays.
In the recent past, oligonucleotides have played an increasing and more pivotal role in diagnostic medicine, forensic medicine and molecular biology research. One primary function of these polymers, in particular, is their use in gene probe assays for the detection of specific nucleic acid sequences.
Gene probe assays are used for a variety of purposes, including: genetic counseling; tissue typing; and molecular biology research. For example, an individual may be tested to see if he or she carries the gene for Huntington's disease or cystic fibrosis. Another use of gene probe assays include determining compatibility prior to tissue transplantation, and for matching up tissue or blood samples for forensic medicine. Finally, in molecular biology research, these assays are extensively employed to explore homologies among genes from different species, and to clone genes for which only a partial nucleic acid or amino acid sequence is known, such as in the polymerase chain reaction (per).
These types of assays typically test for the presence of a specific nucleic acid sequence, usually a DNA sequence, although RNA sequences may be employed as well. As is well known in the field, this is accomplished utilizing oligonucleotides synthesized to have specific, predetermined sequences. The sequence of an oligonucleotide probe may be based on known amino acid sequences, known DNA sequences, or may be a “guess” probe, based on homology to known or putative DNA or amino acid sequences. Since DNA is a “degenerate” code, and several DNA sequences will result in a single amino acid sequence, gene probe assays based on an amino acid sequence frequently utilize pools of related oligonucleotides; each having a different specific sequence of nucleotides. Thus it is often necessary to create a large number of related but distinct oligonucleotides to clone a single gene.
In addition to the actual sequence, there are a number of parameters which may be altered in the synthesis of oligonucleotides. It is also frequently necessary to synthesize these polymers in a variety of lengths, since generally, the longer the oligonucleotide probe, the more specific the gene probe assay will be; specificity may or may not be desired in any particular application. Oligonucleotides may be made of deoxyribonucleotides, or ribonucleotides, or mixtures. Alternatively, oligonucleotides with modified or nonstandard bases or non-radioactive labels incorporated may be desirable. Similarly, oligonucleotides with altered ribose-phosphate backbones may be created for some applications.
There are several other uses of oligonucleotides besides the gene probe assay use. For example, the formation of a single stranded oligonucleotide with a specific sequence may be used in the formation of extremely stable triplex DNA structures. Other uses include direct construction of synthetic genes and plasmid vectotrts by ligating or joining together the component 5′-phosphate oligonucleotides.
Accordingly, as the use of synthetic oligonucleotides has increased, so has its demand. In turn, this has spawned development of new synthesis apparatus and methodologies for basic procedures for custom sequence defined oligonucleotides. These apparatus and methods, however, are generally very expensive to employ and not readily available for mass production thereof. Typically, the present generation automated DNA sequential synthesizers place a derivatized solid support, such as controlled pore glass (CPG), into an individual reaction chamber of a column to provide a stable anchor on which to initiate solid phase synthesis. Using a series of complex valving, and pumps coupled to the column, the appropriate selected reagents are sequentially filtrated through the chamber in a predetermined manner. Contact of the reagent with the polymer units pre-affixed to the CPG, which is retained and supported in the chamber by a sample support porous frit, causes a reaction resulting in sequenced growth thereon.
While each column of this assembly is effective to rapidly mass produce a homogenous population of sequence defined oligonucleotides, the current assemblies only offer four (4) column capabilities. Increased column capacity is limited due to physical limitations of the valving configuration. Hence, only four independent synthesis cycles can be performed simultaneously. Further, since the synthesis apparatus is not generally amenable to integrated automation with other robotic lab instrumentation, an operator must intervene to load and remove each individual synthesis column manually. Such handling increases human error.
A more important limitation is that all the reagents are funneled through a common manifold passage. Only one reagent or combination thereof, thus, can be simultaneously deposited in selected columns. For example, the reagent “tetrazole” cannot be deposited in column one while a particular amidite reagent is simultaneously being deposited in column four. In addition, for each independent synthesis or reaction, the common manifold passage and associated valving must be flushed with a cleansing reagent so that residual amidite or deblocking reagents will not be undesirably deposited in a column. This approach wastes time, as well as increasing operator costs.
Synthesis of arrays of bound oligonucleotides or peptides is also generally known in the art. In one approach to parallel synthesis, known as the Tea-bag?? method or disk design, an array of individual packets or disks of solid support beads are physically sorted into four (4) amidite subsets for treatment with the selected amidite. After each packet of beads has been treated with the common reagent, the packets must again be manually resorted into the four subsets for the subsequent synthesis cycle. Such sorting and resorting becomes too burdensome and labor intensive for the preparation of large arrays of oligonucleotides.
Another approach using arrays is the pin dipping method for parallel oligonucleotide synthesis. Geysen, J. Org. Chem. 56, 6659 (1991). In this method, small amounts of solid support are fused to arrays of solenoid controlled polypropylene pins, which are subsequently dipped into trays of the appropriate reagents. The density of arrays, however, is limited, and the dipping procedure employed is cumbersome in practice.
Disclosed at the Southern, Genome Mapping Sequence Conference, May 1991, Cold Spring Harbour, N.Y., is still another scheme for oligonucleotide array synthesis in which selected areas on a glass plate are physically masked and the desired chemical reaction is carried out on the unmasked portion of the plate. The problem with this method is that it is necessary to remove the old mask and apply a new one after each interaction. Fodor et al., Science 251, 767 (1991) describes another method for synthesizing very dense 50 micron arrays of peptides (and potentially oligonucleotides) using mask-directed photochemical deprotection and synthetic intermediates. This method is limited by the slow rate of photochemical deprotection and by the susceptibility to side reactions (e.g., thymidine dimer formation) in oligonucleotide synthesis. Khrapko et al., FEBS Letters 256, 118 (1989) suggest simplified synthesis and immobilization of multiple oligonucleotides by direct synthesis on a two-dimensional support, using a printer-like device capable of sampling each of the four nucleotides into given dots on the matrix. However, no particulars about how to make or use such a device are provided.
In summary, the related art generally contains numerous ideas and information related to the synthesis of arrays of oligonucleotides or peptides for the determination of nucleotide sequences or the amino acid sequences of specific binding peptides. However, existing or suggested methods are limited, and do not conveniently and reliably mass produce very large arrays necessary for effective large-scale sequencing.
Accordingly, it is an object of the present invention to provide a polymer synthesis apparatus and method for building sequence defined polymer chains.
Another object of the present invention is to provide a polymer synthesis apparatus and method for preparing large quantity arrays of oligonucleotides or peptides in a reproducible and rapid manner.
Yet another object of the present invention is to provide a polymer synthesis apparatus and method which reduces reagent waste during the preparation of large quantity arrays of oligonucleotides.
Still another object of the present invention is to provide a polymer synthesis apparatus and method for preparing large quantity arrays of oligonucleotide at reduced costs.
It is a further object of the present invention to provide a polymer array synthesis apparatus and method which is durable, compact, easy to maintain, has a minimum number of components, is easy to use by unskilled personnel, and is economical to manufacture.
In accordance with the foregoing objects, one embodiment of the present invention provides a polymer synthesis apparatus for building a polymer chain by sequentially adding polymer units in a reagent solution. The synthesis apparatus comprises a head assembly having a plurality of nozzles mounted thereto in generally spaced-apart relation. Each nozzle is coupled to a reservoir of liquid reagent for controlled delivery therethrough. Further, a base assembly is included having at least one reaction well, and a transport mechanism coupled to at least one of the head assembly and the base assembly to produce relative movement therebetween. This positions the reaction well and a selected one nozzle in alignment for deposition of a liquid reagent into the reaction well for synthesis of a polymer chain. A sliding seal is positioned between the head assembly and the base assembly to form a common chamber enclosing both the reaction well and the nozzles therein.
The synthesis apparatus may further include an inlet into the common chamber positioned upstream from the nozzles, and an outlet out of the common chamber positioned downstream from the nozzles. A pressurized gas source is coupled to the inlet for continuously streaming a gas through the common chamber from the chamber upstream to the chamber downstream and out of the outlet to sweep the common chamber of toxic fumes emitted by the reagents, as well as keep air and moisture from seeping in.
In another aspect of the present invention, a polymer synthesis apparatus is provided comprising a base assembly including a reaction well, and having at least one orifice extending into the well. At least one solid support is disposed in the well for growing and immobilizing a polymer chain thereon. Reagent solution in the well is in contact with the solid support and at least one polymer unit of the polymer chain affixed to the solid support. Further, a retaining device is included positioned in the well, and is formed and dimensioned to substantially prevent passage of the solid support through the orifice. The orifice has an entrance into the well and an exit out of the well, and is of a size and dimension to form a capillary liquid seal to retain the reagent solution in the well to enable polymer chain growth therein. To retain the solution in the well, a pressure differential between a first gas pressure exerted on the reaction well and a second gas pressure exerted on the orifice exit must be less than a predetermined amount. Finally, a pressure regulating device is provided for controlling the pressure differential such that upon the pressure differential exceeding the predetermined amount, the reagent solution is expelled from the well through the orifice.
The present invention also includes a method of synthesis of a polymer chain in a synthesis apparatus comprising the steps of: A) aligning the reaction well and a selected one nozzle through the transport mechanism coupled to at least one of the head assembly and the base assembly to produce relative movement therebetween; and B) depositing a liquid reagent into the well from the reagent reservoir through the one nozzle to enable synthesis of a polymer chain. Finally, C) sweeping toxic fumes, emitted by the reagents, from the common chamber through passage of a gas from a pressurized gas source, coupled to an inlet into the common chamber and positioned upstream from the nozzles, and out of the chamber through an outlet out from the common chamber and positioned downstream from the nozzles.
Another method of polymer synthesis is provided for building a polymer chain by sequentially adding polymer units to at least one solid support for growing and immobilizing a polymer chain thereon in a liquid reagent. The method comprises the steps of A) depositing a liquid reagent in the reaction well, having a properly sized orifice, in contact with at least one solid support and at least one polymer unit of the polymer chain affixed to the solid support, and forming a capillary liquid seal to retain the reagent solution in the well to enable polymer chain growth on the solid support. The next step includes B) applying a first gas pressure to the reaction well such that a pressure differential between the first gas pressure and a second gas pressure exerted on an exit of the orifice exceeds a predetermined amount necessary to overcome the capillary liquid seal and expel the reagent solution from the well through the orifice.
The assembly of the present invention has other objects and features of advantage which will be more readily apparent from the following description of the Best Mode of Carrying Out the Invention and the appended claims, when taken in conjunction with the accompanying drawing, in which:
While the present invention will be described with reference to a few specific embodiments, the description is illustrative of the invention and is not to be construed as limiting the invention. Various modifications to the present invention can be made to the preferred embodiments by those skilled in the art without departing from the true spirit and scope of the invention as defined by the appended claims. It will be noted here that for a better understanding, like components are designated by like reference numerals throughout the various figures.
Attention is now directed to
In one embodiment, the synthesis apparatus 20, briefly, comprises a head assembly, generally designated 21, having a plurality of nozzles 22 (FIG. 2} mounted thereto in generally spaced-apart relation. Each nozzle 22 is coupled to a reservoir 23 (
In the preferred embodiment, an array of wells 26 (
This array technique for sequence defined oligonucleotide synthesis first evolved from the high density oligonucleotide array chip assembly for hybridization sequencing disclosed in U.S. patent application Ser. No. 07/754,614, filed Sep. 4, 1991, now pending. Hence, this array technique, alone, is not claimed as a novel feature of the present invention. That assembly, however, is neither suitable nor appropriate for the large-scale production capabilities of the present invention as will be described henceforth.
To simplify organization and manufacture of polymer chains, a delivery assembly 43 (
The delivery assembly 43 of the present invention communicably couples each bank of nozzles to a common reagent reservoir through independent dispensing tubes 44. Each tube includes a passageway 45 (
A distal end of each tube 44, as shown in
There are two important concerns in liquid reagent delivery through nozzles 22: 1) how to eject a droplet cleanly so that a drop is not left hanging on the end of the nozzle; and 2) how to keep the contents of the reaction chamber from splashing when the stream of reagent is delivered into the well. Further, the ejection velocity of the reagent from the nozzle must be sufficient to induce mixing between the first and second delivered reagent in the reaction chamber. Very small droplets can be ejected cleanly at high ejection velocities, but do not have sufficient kinetic energy to overcome the surface tension of the liquid already in the well to cause mixing. In contrast, larger droplets also eject cleanly at high ejection velocities, but tend to splash the contents into adjacent wells. At lower ejection velocities, the reagents tend to leave the last drop hanging from the nozzle tip, which is also a function of the cross-sectional area of the tip. Moreover, the flow rate of liquids through small capillary tubing varies directly with the delivery pressure and inversely with the length of the tube and inversely with the diameter. All these variables must be taken into consideration when developing delivery pressure and nozzle configurations, as well as the materials of construction, so that the reagents can be expelled cleanly without leaving a residual drop of liquid reagent hanging from the nozzle tip. Hence, depending on the liquid reagent, it may be more beneficial to dispense it in a continuous stream, a series of pulses or in droplet form.
Each reagent reservoir 23, as shown in
To assure a constant delivery pressure across each dispensing tube 44, and hence, a constant delivery rate of liquid reagent through any nozzle in a bank or row 40 of nozzles 22, each dispensing tube will independently terminate in the liquid reagent contained in the reagent reservoir. Thus, independent of the number of nozzles set to deliver, this configuration will not suffer uneven rate delivery caused by varying line pressure.
In accordance with the present invention, base assembly 25 and head assembly 21 cooperate with transport mechanism 27 for relative movement between the base assembly and the head assembly to align the array of wells with the array of nozzles at a plurality of positions. Preferably, the transport mechanism moves the base assembly along (phantom lines in
Transport mechanism 27 includes a stepped motor assembly 62, schematically represented in
Before polymer synthesis begins or after the synthesis process has ended, the array of wells may be positioned outside of common chamber 31 and exposed to the open environment for access by moving the wells 26 (via movement of base assembly 25) to the extreme right or left of sliding seal 30. Hence, the well array may be cleaned or loaded with solid support material, to be discussed. Further, the wells and nozzles, once inside the common chamber, may be accessed through a hinge assembly 63, as shown in
Referring now to
Sliding seal 30 must be formed to maintain environmental containment while permitting the base assembly to slide relative the head assembly. In the preferred form, sliding seal 30 is provided by an elastic, rectangular-shaped, hydrofoil or balloon seal gasket having an upper end mounted to head bottom surface 51, and an opposite or lower end 64 in sliding contact with base top surface 52. This special gasket, preferably composed of rubber or the like, increases seal integrity between gasket lower end 64 and base top surface 52 as the pressure in common chamber increases.
To facilitate sliding contact, while maintaining environmental containment, gasket seal 30 includes a stick-free coating or layer 65 (
It will be understood that the seal between gasket lower end 64 and base top surface 52 need not be hermetic. The primary function of the seal gasket is to exclude oxygen from the reaction chamber. Thus, it is important to normally maintain a minimum positive pressure inside common chamber 31 at all times during synthesis which is slightly greater than atmospheric pressure so that the flow of gas, should a leak occur, would be outward. This minimum positive pressure differential is generally about 1/100 psi to about 1/10 psi.
As previously indicated and as viewed in
The gas inlet is preferably provided by an elongated inlet slot 70 (
Because of the gas flow through the chamber, the acid and moisture sensitive phophoramides are positioned upstream from the deblocker and capping reagents, forming a diffusion gas barrier, which maximizes the sweeping effect. Accordingly, in
In another aspect of the present invention, as best viewed in
Briefly, after proper alignment between selected wells 26′ with selected nozzles 22′ (
A retaining device, generally designated 84, is included positioned in the bottom of well 26 between orifice 74 and the solid support 75 which is formed and dimensioned to substantially prevent passage of the solid support through the orifice. Retaining device 84 is preferably provided by a polyethylene or glass fiber frit which acts as a filter membrane permitting the reagent solution to flow therethrough while retaining the solid support and polymer chain grown thereon in the well. Hence, the porosity of the frit is also a factor in the formation of the capillary liquid seal and in the determination of the pressure differential necessary to purge the reaction well.
To regulate and control pressure differential between common chamber 31 and lower catch basin 81, as mentioned, pressure regulating device 82 is provided operably coupled therebetween. In the preferred embodiment, pressure regulating device 82 is integrated with the gas flow assembly employed to flush the head space in common chamber 31 of reagent toxins. Upon the inert gas freely sweeping the chamber from gas inlet 70 to gas outlet 71, the minimum pressure differential is generally retained between about 1/100 psi to about 1/10 psi. This pressure differential which is sufficiently positive to prevent seepage of environmental air into the common chamber, while being insufficient to overcome the capillary forces of the capillary liquid seal in each well. By preventing or restricting the outflow of inert gas through gas outlet 71, the pressure inside chamber 31 can be increased, thereby increasing the pressure differential to purge the wells (
The pressure regulating device 82, hence, includes a chamber valve 85 (
The liquid reaction solution will hot leak out of or be purged from well orifice 74 until there is a sufficient head of liquid in the well or a sufficient gas pressure differential between the common chamber and the lower catch basin to overcome the capillary forces in the orifice. The rate of gravity-driven and pressure-driven leakage from the orifice is primarily governed by the viscosity of the solvent, the porosity of the frit, the size of the orifice, and the gas pressure differential. For instance, a 10μ UHMW polyethylene frit and a 0.015 in2 orifice will support at least 0.79 in. liquid head of acetonitrile (having a viscosity of about 0.345 centipose (7.2×10−5 (lbf·s)/ft2 at an operating temperature of about 68° F.)) before beginning to overcome the capillary forces in the orifice. On the other hand, by increasing the pressure differential above the predetermined amount (generally about 1 psi) to about 5 psi, purging of the well will occur rapidly. In practice, it is necessary to maintain a pressure differential between about 2.5 psi to about 5 psi to sufficiently purge the reaction wells simultaneously of reagent solution. As the individual wells begin to empty, the flow rate of inert gas through the empty wells of the microtiter plate substantially increases which decreases the pressure in common chamber 31. Accordingly, this decrease in interior pressure further decreases the purging or draining rate of the reagent solution through the orifices, an effect magnified by retaining filter membrane 84.
It will be appreciated that the pressure differential may also be created by forming a vacuum in lower catch basin 81 to purge the reaction wells.
To coordinate all the simultaneous functions, a control mechanism 90 (
Oligonucleotides are typically synthesized on solid supports of controlled pored glass (CPG) having the first nucleotide previously linked to the CPG through the 3′-succinate linkage. Hence, upon preparation for polymer synthesis, each well is individually loaded with the correct CPG derivative. While one could begin a full plate of synthesis using only one CPG derivative, for example T (i.e., dT-Icaa-CPG), it is more desirable to perform array synthesis where any base can be in the first position. However, individually weighing and transferring 0.5 mg quantities of the appropriate dry CPG derivative to each well can be tedious and time consuming.
In accordance with the present invention, a balanced density slurry technique is employed to deposit the correct amount of CPG into a reaction well. By suspending the CPG in a suspension solution, a desired amount of CPG can be accurately deposited in a well by pipetting, either automatically or manually, a corresponding volume of suspension solution therein. For example, a non-settling 10%-1% weight/volume suspension of CPG can be prepared in a 2.5:1 volume/volume dibromomethane-dichloromethane solution. Subsequently, the CPG can be washed and purged of suspension solution before synthesis using the technique mentioned above.
In another aspect of the present invention, a method of synthesis of a polymer chain is provided comprising the steps of: A) aligning reaction well 26 and a selected one nozzle 22 of synthesis apparatus 20 through transport mechanism 27 coupled to at least one of head assembly 21 and base assembly 25 to produce relative movement therebetween; and B) depositing a liquid reagent 24 into well 26 from reagent reservoir 23 through the one nozzle to enable synthesis of a polymer chain. Finally, C) sweeping toxic fumes, emitted by the reagents, from common chamber 31 through passage of a gas from a pressurized gas source, coupled to an inlet 70 into common chamber 31 and positioned upstream from the nozzles, and out of the chamber through an outlet 71 out from the common chamber and positioned downstream from the nozzles.
Another method of polymer synthesis is provided for building a polymer chain by sequentially adding polymer units to at least one solid support for growing and immobilizing a polymer chain thereon in a liquid reagent. The method comprises the steps of A) depositing liquid reagent 24 in reaction well 26, having a properly sized orifice 74, in contact with at least one solid support 75 and at least one polymer unit of the polymer chain affixed to solid support 75, and forming a capillary liquid seal to retain the reagent solution in well 26 to enable polymer chain growth on solid support 75. The next step includes B) applying a first gas pressure to reaction chamber 31 such that a pressure differential between the first gas pressure and a second gas pressure exerted on an exit 80 of orifice 74 exceeds a predetermined amount necessary to overcome the capillary liquid seal and expel the reagent solution from well 26 through orifice 74.
By repeating the steps of the two above-mentioned methods, one continuous chain of polymer units can be formed.
The following example serves to more fully describe the manner of using the above-described invention, as well as to set forth the best mode contemplated for carrying out various aspects of the invention. It is to be understood that this example in no way serves to limit the true scope of the invention, but rather are presented for illustrative purposes. It is to be understood that any method of oligonucleotide synthesis may be utilized in the present invention.
Synthesis of an Array of 15 Oligonucleotides
The general synthesis procedure follows published procedures for the phosphoramidite and hydrogen phosphonate methods of oligonucleotide synthesis; for example, the methods outlined in Oligonucleotides and Analogues: A Practical Approach, F. Eckstein, Ed. IRL Press, Oxford University; Oligonucleotide Synthesis: A Practical Approach, Gait, Ed., IRL Press, Washington D.C.; and U.S. Pat. Nos. 4,458,066, 4,500,707 and 5,047,524, all hereby incorporated by reference. It is to be understood that other methods of oligonucleotide synthesis may be used in the present invention.
In general, the basic steps of the synthesis reaction are as follows, with appropriate acetonitrile washing steps:
Accordingly, a sequence file was generated which indicated the 3′-5′ sequence and well number for each oligonucleotide to be synthesized, the scale of the reaction for each nucleotide, and whether a final detritylation was to be performed on the product at the end of the reaction. The software was designed to support simultaneous independent synthesis of oligonucleotides of different lengths and scale. The sequence of the 15 different oligonucleotides is shown in Table 1 below.
AAG TCT TGG ACT TAG AAG CC
GGG TCA CTT CTT TGT TTT CG
CCT TTA CTT TTC GGT CAA GG
TGA CTG GTA GGT CCA CTG CA
ACC TCA CTT CGG TAA CTT AAA
CCA AAT TTT GAG GTA ACC ACT T
GTG CTA TCC GGG ACA CCA
GGA AGA TCC ACG AGT CGT TT
CGT CCA CCT CTT CTT TGA AGA
TGG TAG TAA TCC TAC CGT CT
GCG AAG GTC TAC TCT CGT C
GTT CAG AGT AAG GAG TCG TGG
GTG GAA CCA TAG ACA ACC ACA TAT
TTG TTC ATC TTT GTG TAG GGT AC
AGA AAA GTC ACG GTC ACA CT
A command file was generated which contained the sequence of reaction steps performed during a coupling cycle. The basic commands were WASH (volume), DEBLOCK (volume), COUPLE, CAP, OXIDIZE, WAIT (seconds) and DRAIN (pressure/vacuum seconds), as shown in Table 2 below:
A configuration file was generated which contained the operating characteristics of the machine, the liquid flow rates, the concentration and molar equivalents used for each of the reagents, as shown in Table 3 below.
96 well plates were used which have a conical lower section which has been recessed to receive a pressed-fit 0.196 inch filter disk. Below the filter was the flow restriction and capillary liquid seal orifice, 0.020 inch diameter×0.350 inch length. The outside diameter of the capillary seal at the exit tip was 0.035 inch. When fitted with Whatman GF/A filters, the gas flow rate through a completely dry plate at an upper chamber pressure of 1 psi was 10 liters per minute. At 0.02 psi upper chamber pressure, the capillary seal at the bottom of the synthesis well supported an acetonitrile liquid head of approximately 1 cm above the filter before slow leakage commenced. The plates were fabricated of molded polypropylene.
To each well was added the correct amount of CPG with derivatized first base, either A, T, C, or G. For example, 0.5 mg CPG was used in a 20 nanomole scale reaction. The CPG beads were added as a slurry in dibromomethane-chloroform pipetted into each well. This step was performed in ordinary lab atmosphere prior to loading the plate into the machine. After loading the plate into the machine, a WASH acetonitrile cycle was done to rinse the slurry, and to verify that all wells were draining properly.
The nozzle valve reservoirs were flushed with argon and then filled with fresh reagents from the supply bottles by pressurized transfer to avoid air and water contamination. The valve reservoirs were then pressurized to the configuration pressure (4 psi). The nozzle tubing was then primed by dispensing a small amount of liquid with the sliding plate in the purge position for each reagent.
During reagent addition and reaction wait periods, the laminar gas sweep flow across the reaction chamber was regulated to 0.4 liter per minutes with an internal pressure of approximately 0.02 psi. During spent reagent removal and washing cycles, the chamber pressure was increased to a maximum of 5 psi. At 2.5 psi, the synthesis wells drained at a rate of approximately 150 λ/sec.
The machine was then switched into AUTOmatic mode in order to commence actual synthesis. The machine paused during the first, second and last cycles in order to allow collection of the DEBLOCK trityl products for colorimetric analysis. This was accomplished by sliding a second 96-well microtiter plate into the lower vacuum/drain chamber, immediately beneath the capillary exit tips of the synthesis plate. The optical density of detritylation solution at 490 nanometers was read using a plate reader. The value for the first cycle confirmed the amount of CPG loaded into a well, and comparison of the second cycle with the last cycle permitted calculation of the synthesis coupling efficiency. The final cycle value also indicated the amount of oligonucleotide which will be obtained after sidechain deprotection by ammonia cleavage.
After the machine completed the synthesis in all the wells, the synthesis plate was removed from the machine. The synthesis plate was stacked on top of a second 96 well deprotection plate (Beckman deep-well titer plate 267001). The oligonucleotides were cleaved from the support by pipetting 200 microliters of concentrated NH4OH into each well followed by incubation at 25° C. for 15 minutes. The ammonium cleavage solutions were eluted through the filter and capillary exit into the wells of the deprotection plate by pressure, vacuum or preferably brief centrifugation of the stack. This cleavage step was repeated twice with fresh aliquots of ammonia.
If the phosphoramidites used for synthesis had benzoyl and isobutyryl protecting groups, the final deprotection was completed by heating the crude ammonia cleavage product. The wells of the deprotection titer plate were sealed with a dimpled silicon rubber cap (Beckman 267002), and the caps held in place using a spring-loaded plate press. The sealed apparatus was heated in an air oven or water bath at 55° C. for 8-15 hours, and then cooled in an ice bath. The deprotection plate was removed from the press and centrifuged briefly prior to removing the cap.
The ammonia solution was then evaporated to dryness using a Savant 210 Speed Vac equipped with a microplate rotor. The oligonucleotide pellets thus obtained were generally sufficiently pure for use directly in most sequencing primer and PCR applications. The amount of oligonucleotide predicted by the last trityl assay was verified by optical density at 260 nanometers, and the homogeneity of the product assayed by HPLC or capillary gel electrophoresis. Capillary electrophoresis analysis was performed on an Applied Biosystems 270A instrument according to the manufacturer's protocois for oligonucleotides 14 and 15 from the above run are shown in
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|Clasificación de EE.UU.||435/283.1, 435/288.3, 422/503, 422/502, 536/25.3, 536/23.1, 435/288.5, 422/501, 422/515|
|Clasificación internacional||C12M1/00, B01L3/02, B01J19/00, C08F2/04, C40B50/14, C12N15/09, C08F2/00, C40B40/06, C07K1/02, C07H21/00, C08G85/00, C07H21/04, C07K1/04, G01N35/10, C40B40/10, C40B60/14, C07B61/00, C07H21/02|
|Clasificación cooperativa||B01J2219/00416, C40B60/14, C07K1/04, B01J2219/0072, B01J2219/00418, B01J2219/00689, C40B50/14, B01J2219/00328, C07B2200/11, B01J2219/00315, B01J2219/00722, B01J2219/00725, B01J2219/00313, B01J2219/0059, B01J2219/00585, B01J2219/00695, C07K1/045, B01J2219/00286, B01J2219/00686, B01J2219/00459, B01J2219/00454, B01J2219/00414, C40B40/06, B01J2219/00423, B01J2219/00364, B01J2219/00596, C40B40/10, B01J2219/0031, C07H21/00, B01J2219/005, B01J2219/00369, G01N35/1072, B01J19/0046|
|Clasificación europea||B01J19/00C, C07K1/04, G01N35/10M3, C07K1/04B, C07H21/00|
|10 Abr 2012||CC||Certificate of correction|
|7 Ago 2015||REMI||Maintenance fee reminder mailed|
|27 Dic 2015||LAPS||Lapse for failure to pay maintenance fees|
|16 Feb 2016||FP||Expired due to failure to pay maintenance fee|
Effective date: 20151227


Tuesday night was a vastly different night for both the Boston Bruins and Edmonton Oilers. The Oilers claimed a huge 4-2 victory at home that pushed them to second in the Pacific Division. The Bruins, meanwhile, lost 3-0 in Columbus just 24 hours after blowing a 5-2 lead in Philadelphia.
To make matters worse for the Bruins, goalie Tuukka Rask was injured on a dirty play just under two minutes into the game. Rask took an elbow to the head from Blue Jackets forward Emil Bemstrom. Rask suffered a concussion, according to coach Bruce Cassidy.
Bruins fans are hot this morning, understandably so. If two lackluster performances against lesser teams isn’t enough to fire you up, then your All-Star goalie being taken out by a cheap shot should be. The response from the Bruins sent the city of Boston into an inferno.
Why? Mostly because there really was no response. The team formerly known as the “Big, Bad Bruins” stood by and watched after the filthy elbow. There was no response, there was no accountability for Bemstrom. In a city that loves its old school hockey, this is unacceptable.
Brett Ritchie, the 6’3″ power forward signed July 1st, stood by and didn’t really offer a response. Ritchie is already on thin ice with the club, and not providing the grit he was expected to bring probably won’t help his case.
Bruins fans are clamoring for an enforcer to come help this team protect its star players.
Why do I share this with fans of the Oilers? Edmonton might have just the player for the Bruins.
Jujhar Khaira is 6’4″ and 212 pounds. He’s a big boy that isn’t afraid to drop the gloves and who has a knack for playing physical, sometimes even dirty. Khaira is the modern enforcer. He can play the game a bit, but can still police the ice and intimidate the opposition.
Khaira has fallen out of favor in Edmonton this season. It is his second consecutive underwhelming season. The offense just isn’t there for Khaira, and most nights he is a drag on possession and scoring.
At times, he has been a liability for a team that can’t afford to have any in the lineup.
Khaira’s fancy stats are not great. His Corsi For percentage of 44.5% is below the team average. Edmonton is not a great five-on-five team, but Khaira has sunk even further than the average Oilers player this season (-3.2% CF% rel)
His 45.6% Fenwick For % is also below the team average (-2.9% rel), while his expected goals for of 9.9 at five-on-five is one of the worst marks on the team. It is also well below his established levels.
Khaira’s six goals aren’t far off, but the assists are all but dried up. He has just two this season after collecting 15 a season ago and ten in 2017-18. He’s on pace for less than five helpers this season.
At five-on-five, it has been a disaster of a season for Khaira. That doesn’t mean he has no uses. He is one of the better penalty killers for the club, and does display some strong two-way acumen. His physical presence matters to teams, especially one like the Bruins that is planning on a deep playoff run.
Why Would The Oilers Deal Khaira?
The Edmonton Oilers are slowly building a team that they hope will contend for a Stanley Cup. In that process, the club has discovered a solid bottom six forward in Joakim Nygard that does a lot of the things that Khaira does. In addition, Tyler Benson is close, if not ready, for NHL duty.
Khaira has one more season with a cap hit of $1,200,000 remaining. It isn’t a major number, but it is an overpay for a player who likely should be relegated to the pressbox right now.
The Oilers will need every penny they can find this summer, and will need room on their roster for players like Benson to graduate to the NHL and to make some meaningful additions to the roster this summer.
Benson will make his NHL debut sooner rather than later. If, like Kailer Yamamoto has done, he can find a way to stick, the Oilers will need to make room for him. Khaira is having his second straight disappointing season, but does have value as a depth player and penalty killer.
A team like the Bruins needs a jolt right now. They also need a player who can bring some of the physical intimidation that they are lacking. Khaira can do that, replacing someone like Ritchie or veteran David Backes in the lineup.
The Oilers likely would settle for a mid-round pick or even a prospect to get Khaira off the books and to open up a roster spot. Would the Bruins even have interest? Don Sweeney needs to act sooner rather than later, and Khaira could be a low risk addition that adds some of the missing ingredients in Boston.


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Stumbras Premium Organic Vodka
is produced from wheat grown on an organic farm and deep subsurface water. Its production also includes filtration through a unique linen filter – the very unique filter in the world. Though no longer grown in Lithuania, flax is still a distinctly Lithuanian plant and thus acted as the leading design inspiration. The natural, blue colour of the flax blossom, the pattern on the bottle neck reminiscent of mature flax, and the linen filter symbol are the main eye-catching design elements, while the paper used for the label, the subtle foil decor and the embossed details evoke sensations of touch. Stumbras Premium Organic Vodka is clear, crisp with a smooth, pleasant finish, and is an incredible mixer for almost any cocktail recipe.

domain: artistic


Domestos changes behaviours
‘Cleaner Toilets, Brighter Future’
In an effort to support the South African Governments service delivery objectives to raise awareness of the importance of functioning and hygienic toilet facilities, Unilever’s Domestos brand launched the Cleaner Toilets, Brighter Future’ Campaign in 2010.
The initiative, which forms part of Unilever’s Sustainable Living Plan, has helped to renovate 26 schools, benefitting 26 000 children, by providing access to clean, safe and germ free bathrooms. These schools were refurbished with newly installed toilets, sinks and taps; and freshly painted walls and doors.
Furthermore, the ‘Germ Buster Club’ - a hygiene awareness programme has reached 1360 schools and 1.1m learners since 2015. Running for the third year in primary schools, the project has empowered learners to become brand ambassadors to help spread the important message of keeping toilets clean and hygienic at all time to their families and communities.
Globally, Domestos has announced its target of helping 25 million people gain improved access to a toilet by 2020. In South Africa, the brand aims to improve sanitation in schools and provide better conditions for approximately 1.4 million learners by 2020.
In doing so, Unilever’s global alliance with UNICEF aims to improve sanitation in developing countries through UNICEF's Community Approaches to Total Sanitation (CATS) initiative - a behaviour change programme that promotes good hygiene practices, helps create demand for access to toilets, and raises awareness of the sanitation crisis.
Highlighting a need for ‘Collective Action’, Unilever annually celebrates World Toilet Day on 19 November - an initiative introduced by key global influencers to raise awareness and improve toilet and sanitation conditions worldwide. Through World Toilet Day, Domestos aims to promote Unilever’s vision to help create a brighter future for all South Africans.


FREE NEXT DAY DELIVERY ON UK ORDERS OVER £30. ORDER BY 20TH DEC FOR DELIVERY BEFORE CHRISTMAS!
Gentlemen's Hardware Tool Kit
A mini work bench in a box! The Handy Man Tool Kit in a Wooden Box brings together all the fixing-up necessities for a DIY enthusiast. The solid wood case, with neat handle and traditional latch fastenings, holds a hammer, tape measure, wooden ruler, pliers, multi-ratchet screwdriver and, of course, a pencil! Each premium-quality piece is strong, ergonomic and made to last. Box measures 55 x 345 x 180mm.


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- What can I drink besides water while pregnant?
- Can I drink fresh fruit juice while pregnant?
- Is Orange Juice Good for pregnancy?
- Is Juicing healthy for pregnancy?
- Can baby feel when I rub my belly?
- What are the fruits to avoid during pregnancy?
- Is carrot juice good in pregnancy?
- Is beetroot juice good for pregnant ladies?
- Which fruit is good for pregnancy?
- Is drinking too much orange juice bad during pregnancy?
- Can I drink Coke while pregnant?
- Is pineapple juice good for pregnant?
- Which orange juice is good for pregnancy?
What can I drink besides water while pregnant?
Can I replace water with other fluids?Yes, when it comes to meeting your fluid needs, there are several alternatives to water:Milk.
Milk is a highly recommended drink for mothers-to-be because it is rich in calcium and protein.
Fresh fruit juice.
Soups and broths.
Can I drink fresh fruit juice while pregnant?
No. Unpasteurized juice may contain harmful bacteria from the raw fruits and vegetables used to make the juice. These bacteria can cause food poisoning (such as listeriosis and toxoplasmosis), which can be especially dangerous during pregnancy.
Is Orange Juice Good for pregnancy?
Orange juice is good to drink while you’re pregnant, but you should only have it when it is pasteurized and fortified with calcium. This is especially good to consume if you have high blood pressure or your obstetrician has told you that you are at risk for it—also known as preeclampsia—during your pregnancy.
Is Juicing healthy for pregnancy?
Raw and even cooked vegetables can be especially off-putting during the first trimester of pregnancy. The easiest way to pack a plethora of vitamins and nutrients into a single glass is through juicing. Plus, there’s nothing for the digestive system to break down!
Can baby feel when I rub my belly?
By now, you have probably already experienced the sensation of your baby kicking and turning inside you. He or she can feel you as well. As you rub your belly, you may be able to identify parts of your baby. A long smooth area is likely to be the fetus’s back.
What are the fruits to avoid during pregnancy?
Fruits that should be avoided during pregnancyPineapple. Pregnant women are advised to stay away from this fruit. … Grapes. Grapes are categorised as one of the most nutritious fruits but many people have raised the question on their effect on pregnant women. … Papaya. Raw papayas are known to lead to miscarriages if consumed by pregnant women. … Watermelons. … Peach.
Is carrot juice good in pregnancy?
7. Healthier pregnancy. Drinking carrot juice is also beneficial during and after pregnancy because it’s full of calcium, folate, potassium, magnesium, and of course vitamin A. Calcium helps your fetus develop strong bones and cartilage, while folate prevents birth defects.
Is beetroot juice good for pregnant ladies?
Cooked, juiced or even raw, beetroot is highly advised to be consumed by pregnant women. This delicious vegetable is packed with numerous nutrients that can protect both you and the baby during this period.
Which fruit is good for pregnancy?
Good Fruits for PregnancyApples. Apples are very high in fiber, which can help regulate a woman’s digestion and prevent hemorrhoids – a common issue that many pregnant women face.Citrus. Citrus fruits like lemons and oranges are chock full of vitamin C. … Bananas. … Kiwis. … Watermelon. … Berries.
Is drinking too much orange juice bad during pregnancy?
Drinking orange juice while pregnant makes a healthy addition to your diet as long as you’re drinking juice that’s been pasteurized. Unpasteurized orange juice, or any other unpasteurized juice for that matter, could potentially contain harmful bacteria that may affect your health or the health of your baby.
Can I drink Coke while pregnant?
Yes. The Food Standards Agency recommends that pregnant women shouldn’t take more than 200mg of caffeine a day. A can of Coca‑Cola Classic contains 32mg of caffeine and a can of Diet Coke contains 42mg.
Is pineapple juice good for pregnant?
Pineapple is a safe, healthy choice during pregnancy. Someone might have told you to avoid this fruit because it may cause early miscarriage or bring on labor. However, this is just a myth. There’s no scientific evidence to support that pineapple is dangerous during pregnancy.
Which orange juice is good for pregnancy?
These healthy snacks will help provide plenty of important nutrients for pregnancy, including vitamic C, folic acid and calcium. Not only is this juice high in vitamin C and folic acid, it’s also a good source of potassium, which has been shown to help lower high blood pressure, a particular danger during pregnancy.

domain: biology


Scientists are studying how to tap the energy naturally created by people's bodies—such as heat, sound and movement—to power medical devices without the need to change batteries.
The development, still years from becoming a reality, could spare some of the millions of people with implanted devices like pacemakers from undergoing surgery to replace rundown batteries. Other products, including hearing aids, insulin pumps and pain-management devices, could be made to function without changing batteries, or at least sharply extend their power time. Harnessing the body's energy also could spur development of innovative medical technologies that could potentially monitor the body's inner workings.
In an experiment this month, researchers from Massachusetts Institute of Technology and Harvard Medical School demonstrated in guinea pigs that it is possible to use energy produced by the inner ear, which is needed for hearing, to power tiny sensors without interfering with the animals' functioning. The researchers say the device, which resembles a semiconductor chip, is small enough to be implanted in the ear one day to monitor problems such as ear infections and hearing loss.
Scientists at other labs are trying to capture and convert energy from heart beats, blood flow, lung contractions and arm and leg movements.
Researchers compare the futuristic devices to solar-powered calculators, which work as long as there is sunlight. Some experts expect the first medical devices that tap the body's energy—known as bioenergy harvesting—could be available within a decade.
Pacemaker batteries typically last for five to 10 years before they wear out and need to be replaced with a new battery using surgery. Currently, there is no way to recharge the pacemaker battery once it's depleted. In the future, the hope is the battery will be continually powered by the energy from one's own heart beat. The ultimate goal "is what I call the perpetual device," says Gene Frantz, an expert in power dissipation and an engineer at semiconductor design and manufacturer Texas Instruments Inc. in Dallas. "It doesn't need to be plugged in or operated with" an external battery, he says. Dr. Frantz studies innovation in integrated circuits, including work from many academics and companies researching bioenergy harvesting.
The task is challenging on a number of fronts. Many biological sources of energy tend to emit very low power and often inconsistently, so the energy needs to be captured and stored, such as with batteries, so it can be used by the device, say researchers. Also, energy storage units have to be limited in size if they're going to fit within the body.
An additional hurdle: the devices must not siphon off too much energy, so as to preserve normal body function, says Paul Kohl, a Georgia Tech professor and director of a center that funds researchers, including the MIT group, with grants from the Semiconductor Research Corp., a technology research consortium.
In the work published earlier this month online in the journal Nature Biotechnology, Konstantina Stankovic, an ear surgeon, teamed up with MIT engineering professor Anantha Chandrakasan to figure out a way to tap a reservoir of energy produced by the inner ear to power a medical device. Scientists have been aware of this source of energy, which we need to use in order to hear properly, for some 60 years, but didn't know how to access it.
The researchers designed tiny electrode sensors that could be implanted in the inner ear and would draw just a small bit of current while maintaining normal hearing, says Dr. Chandrakasan, head of MIT's department of electrical engineering and computer science.
It was an engineering feat to design such an ultra-low-power device considering that the ear produces just 70 to 100 millivolts—not even enough juice to power a conventional electronic circuit like a sensor or Wi-Fi chip. A double-A battery generates more than 10 times that amount of voltage, says Dr. Chandrakasan.
The engineers found a way to kick-start a radio-frequency device wirelessly using an external power source initially, but running the device afterward on the output of power generated by the inner ear. To reduce the need for power, they designed a device to wake up and run only when it was taking measurements from the ear.
Dr. Stankovic, an otologic surgeon at the Massachusetts Eye and Ear Infirmary, and a colleague implanted the electrodes into the inner-ear reservoirs of guinea pigs and connected them to the chip outside the animals. The chip, in turn, wirelessly powered a radio-wave device that was located a meter away and measured energy from the ear.
The scientists showed this biological battery was indeed able to be run the radio device and detect the inner-ear environment for about five hours without compromising the guinea pigs' ability to hear. The device is small enough to implant into the middle ear with electrodes connected to the inner ear.
The most immediate application for the device is sensing the inner-ear environment and its vicinity, which can be important to monitor for infection in people with ear disease or a cochlear implant, according to Dr. Stankovic.
Eventually, there may be the possibility that people with some hearing loss could be fitted with these devices to monitor their hearing-loss progression. This could let doctors intervene earlier and possibly help them understand why people lose hearing. The sensors could be used to measure treatment success with those receiving experimental therapies to regrow cells that restore hearing or to deliver drugs.
A University of Michigan team is working on harvesting energy from heart beats to power a pacemaker. M. Amin Karami, an aerospace engineering research fellow, presented data at the American Heart Association annual conference in November showing an experimental energy harvester can convert the energy from the beating of a heart into enough usable electrical energy to, in theory, operate a pacemaker. The harvester hasn't yet been implanted in animals or humans yet.
Their team showed that the device could generate 18 times the amount of energy needed to power a pacemaker using a normal heart beat, according to Dr. Karami. The group measured the vibrations emitted in the chests of pigs and sheep during surgeries, and then used those calculations to estimate how much electrical energy would be generated by the harvester.
"Our next step is to fabricate a complete self-powered pacemaker and examine it with animal tests," wrote Dr. Karami in an email.
At Virginia Polytechnic Institute and State University, electrical engineering professor Dong Ha and his team are in the early stages of studying how to employ body heat to power a circuit. Energy is generated by the body's adjustments to differences in temperature between the skin and the outside air, which can be harvested. If successful, such a device could be implanted just under the skin, or even worn in a jacket, to power a device, says Dr. Ha.
Write to Shirley S. Wang at email@example.com


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CBS Goes a Step Further with Coverage of Tight Senate Race
This year, Democrats are close to retaking the Senate. One network’s coverage of the Senate race has gone a step further than the other two.
On Oct. 26, CBS aired a nearly two-minute story on the Indiana Senate race.
The piece followed Democrat and former senator Evan Bayh’s attempt to replace Republican Sen. Todd Young. It also discussed how Donald Trump and Hillary Clinton are impacting this race.
The night before, CBS’s Julianna Goldman had covered the Senate race in Pennsylvania. Again, the story was nearly two minutes long.
Goldman examined the battle between Republican Pat Toomey and Democrat Katie McGinty. She also connected this race to the race for the White House.
ABC and NBC did not cover Senate races either night.
In New Hampshire that week, Hillary Clinton appeared alongside Democratic Gov. Maggie Hassan. All three networks referenced down-ballot races in their Clinton stories for that night.
But only CBS continued extensive Senate race coverage for the next few days.
Are these Senate races worth this kind of network evening news coverage?
Does focusing on any single Senate race tell the whole story? Or would one or two show the bigger picture?
Carol Marin, a political editor for NBC5 in Chicago, said, “It doesn’t have to tell the whole story. It doesn’t have to carry the full importance of a total election.”
She praised CBS’s pieces on individual Senate races.
“My vote is a very affirmative ‘have at it and do it’. We should know who these people are and we should know what the big contests are. I’d rather see that than endless hurricane stories.”
But she added, “There isn’t a right or wrong. There isn’t an absolute value here. It’s a producer’s call. A newscast is like a canvas… and you’re painting a picture of the day. On that canvas you have a lot of different nuances and varieties and textures.”
Marin also said there’s a lesson for viewers here.
“This is why you don’t stick with one news outlet. Each of them gives you a different way of looking at it.”

domain: politics


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The Egyptian aviation authority announced that the Russian “Nordwild” company will make 4 flights a week, starting from next March 28, to Sharm El-Sheikh and Hurghada.
The Egyptian Aviation Authority stated that the trips will be two flights to Hurghada, and two flights to Sharm El-Sheikh, after the approval of the Russian authorities.
And the head of the Egyptian Civil Aviation Authority, Ashraf Noyer, announced that the Russian company had applied to the authority to operate its flights on March 28, and would be directly from Russian airports to Sharm El-Sheikh and Hurghada, and the flights would be increased as soon as the regular operation.
Noir pointed out that a Russian delegation consisting of 17 members of the security inspectors of the Russian Ministry of Transport recently undertook an inspection tour, which lasted 7 days, at Sharm el-Sheikh and Hurghada airports, and they praised the security measures followed at the two airports.
The Russian authorities had returned EgyptAir and Airflot flights last year between Moscow and Cairo only, on the condition that the Russian passengers did not go to Hurghada and Sharm el-Sheikh by flying and heading only by land.
The return of the “Nordwild” company is the first Russian tourism company that will travel to Sharm El Sheikh and Hurghada, after the flight stopped since the accident of the Russian plane crash on October 31, 2015 in Sinai.
Source: The Seventh Day

domain: travel


Ahead of its weekly videogame sales rankings, GfK Chart Track told MCV that Black Ops sold more than 1.4 million copies during its first 24 hours on UK shelves, generating 58 million euros (nearly $80 million U.S. dollars) in revenue. That take topped the previous record held by Modern Warfare 2.
The latest record follows Activision's announcement yesterday that Black Ops enjoyed the biggest entertainment opening of all time, selling 5.6 million copies and raking in $360 million in 24 hours. And according to GameSpy's own completely unscientific and unsupported data, Black Ops has also set the record for most patched game during its launch week.
Treyarch confirmed via its community forums the various fixes that are now live, as well as the numerous patches that are on the way for the PC, PlayStation 3, and Xbox 360 versions of the game.
"We are listening to you and we've been busy working on updates based on feedback from the community," Treyarch wrote. "We have already deployed a number of updates, and there will be many more to come."
The rundown on live and upcoming fixes for Black Ops:
Live PC Fixes:
-- GameServers.com has bumped sv_maxrate to 25000 across all servers to reduce lag.
-- GameServers.com continues to improve official server distribution in various worldwide regions.
-- Friends fix for join in progress and Friends tab in server browser.
-- Performance improvement for dual and quad core.
-- Improved connectivity with Black Ops Rcon tool.
PC Fixes Coming Soon:
-- GPU hitching on certain graphics cards.
-- Incomplete server browser results.
-- Improvements to Quickmatch joins (results with better ping and reduced lag).
-- Zombies "The game session is no longer available" error.
Live 360 and PS3 Fixes:
-- Villa: User can clip into rock at west edge of grid zone B2 and see through to the other side.
-- Crisis: User can clip into LVT between grid zone A3 and B3 and see through to the other side.
-- User can get an infinite number of Sam Turret, Care Package and Sentry Gun killstreaks (commonly referred to as the "care package glitch.")
-- Radar sweep when there are only two players left in One in the Chamber will stop functioning if the two players have multiple lives when the sweep starts
-- Crash when viewing content (films, clips, etc) in the Community & Recent Games menu
-- Users' rank and stats can get wiped under very specific conditions
-- Online number counts were not updating properly; these are now reporting exact online numbers
-- Parties getting disbanded when host did not find a match quickly
Upcoming 360 and PS3 fixes:
-- Improved matchmaking to find matches faster
-- Improved match and host selection; will result in much better networking conditions for all games (a.k.a., decreased lag)
-- Weekly and Monthly leaderboards stats reporting incorrectly for kills, deaths and assists (All Time leaderboard not affected by this)
-- Additional improvements to party system
Sharkey says: The record-setting launch is a blessing and a curse. It's a curse because so many millions of gamers are playing Black Ops, bugs and glitches will be exploited and talked about ad nauseam. It's a blessing because Treyarch is listening and using the feedback to correct its errors and make a better game.


Country singer Blake Shelton has been named as the fourth and final musician coach of NBC’s upcoming singing competition series The Voice, which premieres April 26. He joins previously announced coaches Christina Aguilera, Cee Lo Green and Adam Levine. With the latest addition, all major genres of popular music are represented on the panel of coaches.
Country Singer Blake Shelton Rounds Out 'The Voice' Team Of Coaches
What's Hot on Deadline
Skirball Fire: Officials Determine Cause Of Bel-Air Blaze; Thomas Fire Still Unchecked; Apple Donates $1M - Update
Keaton Jones Facebook Video Gone After Mom's Confederate Flag Photos Surface: Is Hollywood Still "KeatonStrong"?
Latest TV News
- T-Mobile Acquires Layer3 TV, Plans To Roll Out Pay-TV Bundle In 2018
- Donald Trump Accusers Score Ratings Boost For ‘Megyn Kelly Today’
- Comedy Central Picks Up Off-Network Syndication Rights To ‘The Office’
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- SAG Awards Nominations: The Scorecards
- ‘Mr. Robot’ Renewed For Season 4 By USA Network


The Alpine City Council will hold a special city council meeting, Tuesday, July 8 at 3:30 pm in the City Council Chambers at 803 West Holland Avenue.
The meeting agenda includes a workshop to discuss wastewater improvements (sewer problems and the solution). The workshop will be accompanied by a brief statement of facts, including where the funds for the project will come from.
The meeting will include citizen comments with a time limit of three minutes.
The public is encouraged to attend.


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Cointelegraph By Samuel Haig
The United Nations has accused the North Korean state of stealing $281 million worth of crypto from an exchange during September 2020.
According to Reuters, the findings from a “confidential report” authored by independent sanctions monitors for U.N. Security Council members “strongly suggests” links between the hack’s perpetrators and the North Korean regime. Reuters quoted the report:
“Preliminary analysis, based on the attack vectors and subsequent efforts to launder the illicit proceeds, strongly suggests links to the DPRK.”
Reuters noted the U.N. report accuses North Korea of using the stolen funds to support its nuclear and ballistic missile programs in violation of international sanctions.
Despite the report containing few concrete details surrounding the victimized exchange, crypto data firm Whale Alert’s Frank van Weert speculated the report must be referring to the September 2020 KuCoin hack. Chainalysis estimated losses from the KuCoin hack at $275 million.
The incident saw swift reactions from Tether and major crypto exchanges to quickly freeze crypto assets associated with the attack, preventing roughly 22% of breached funds from being stolen. KuCoin says it has since recovered 80% of the stolen funds.
Citing sources familiar with the incident, the U.N. report cites asserts the attackers breached the exchange by exploiting the smart contracts of DeFi protocols:
“The attackers exploited ‘DeFi’ protocols — i.e., smart contracts that facilitate automated transactions.”
The report estimates North Korea generated roughly $2 billion in total through “widespread and increasingly sophisticated” cyber attacks targeting banks and crypto exchanges as of 2019.
The U.N. notes that one member state believes North Korea stole $316.3M worth of digital assets between 2019 and November 2020, suggesting the regime was responsible for a further $36 million worth of crypto thefts on top of the KuCoin hack during that time.
Last month, a U.S. federal judge ruled that an Ethereum developer accused of helping North Korea to evade sanctions by delivering a speech at a 2019 crypto conference in Pyongyang must soon face a jury. The developer, Virgil Griffith, is accused of conspiring to violate international sanctions by providing “services” to the rogue state.
ICON (ICX) unaffected by South Korean tax investigation into ICONLOOP, says chairman
Cointelegraph By Greg Thomson
The ICON Foundation, creator of the ICON (ICX) project, has issued an official response to the news that South Korean tax authorities are investigating its technical partner, ICONLOOP.
In a statement published on Monday, the chairman of the ICON Foundation, Min Kim, said that the ICON Foundation would not be affected by the investigation, noting that ICONLOOP is operated as a separate entity.
The ICON Foundation is a non-profit organization based in Switzerland. In late 2017 it conducted an initial coin offering for the native coin of the ICON blockchain, ICX, raising over $42 million in the process.
ICONLOOP is the technical partner of the ICON Foundation, operating out of South Korea, and creator of the “loopchain” technology upon which ICON is based. It has previously been employed by the Seoul government in various blockchain pilots, including explorations into COVID-19 tracking apps, citizen rewards and mobile driving licenses.
But past relationships apparently counted for very little at the end of February, when the Seoul Regional Tax Office dispatched 10 employees to ICONLOOP’s headquarters in Euljiro. The investigation is reportedly under way, according to a local outlet article, Paxnet, from March. 8. Such investigations are said to typically last 3 months, with the final judgement expected to be revealed in June.
ICONLOOP is the second blockchain firm to be investigated by Korean tax authorities this year. In January it launched an investigation into the HN Group — issuer of the HDAC token.
Official tax laws for the issuance of virtual assets in South Korea are expected to be implemented next year. In the meantime, the National Tax Service has initiated tax evasion and accounting audits for large domestic virtual asset issuers.
Specifically, the tax office is investigating possible tax evasion that occurs when virtual assets issued by overseas foundations are transferred into South Korea. ICONLOOP is under investigation regarding the possible transfer of the Ethereum (ETH) funds raised by the ICON Foundation in its 2017 ICO, the details of which have not yet been disclosed to authorities. ICONLOOP reportedly argues that it doesn’t need to disclose the details of such transfers, since it operates as a small or medium-sized business, reports Paxnet.
In Monday’s official statement, ICON’s Min Kim, emphasized the legal and jurisdictional distance between the foundation and ICONLOOP, saying:
“We want to make it clear that the ICON Foundation is not under investigation by the Korean National Tax Service authorities. The ICON Foundation is regulated by the Swiss Financial Market Supervisory Authority (FINMA).”
Kim said the investigation would not affect the ICON Foundation, or the ICON project at large, adding that all legal requirements have been met. Kim said: “ICONLOOP is operated independently from the ICON Foundation. ICONLOOP is an important technical partner for the ICON Foundation.” He added further:
“We are confident that this investigation would not affect the deliverables and progress for the ICON project.”
Cointelegraph sought further comment and clarification from the ICON Foundation and ICONLOOP. This article will be updated should they reply.
Finance Minister predicts “very calibrated” stance
Cointelegraph By Andrew Thurman
Yet another “crypto ban” turns out to be temporary FUD.
In an interview with CNBC this morning, Indian Finance Minister Nirmala Sitharaman said that reports of a blanket ban on cryptocurrencies are overstated. While negotiations are ongoing, she said she expects the end result to be more tempered:
“Yes, a lot of negotiations, discussions are happening, with Reserve Bank,” said Sitharaman. “Obviously the Reserve Bank will be taking a quorum on how, what kind of unofficial currency, cryptocurrency will have to be planned, and how it has to be regulated. But also, we want to make sure that there’s a window available for all kinds of experiments which will have to take place in the crypto world.”
She went on to say that regulations won’t be as “severe” as have been previously reported. Authorities will “look inward” and take a “very calibrated” stance, in contrast to the “mixed messages coming in from across the world.”
“The world is moving fast with technology. We can’t pretend that we don’t want it. […] I can only give you this clue: that we are not closing our minds, we are certainly looking at ways in which experimentations can happen in the digital world, in cryptocurrency and so on.”
— BlockchainedIndia (@blockchainedind) March 6, 2021
The comments from Sitharaman is no doubt a source of relief for crypto businesses, users, and hodlers in the world’s second most populous country. Earlier this month, a report from Bloomberg citing a senior Indian financial minister said that the country would be banning all cryptocurrencies.
The hypothetical ban drew widespread criticism from across the crypto community, with some likening it to an attempt to ban the Internet. Some companies found the reports to be hot air, however, and continued on with developments apace.
The United Arab Emirates’ green digitization vision
Cointelegraph By Selva Ozelli
The United Arab Emirates is the world’s sixth-largest oil producer and one of the richest countries in the world, with a gross domestic product per capita of above $43,000 as of 2019, according to the World Bank. As per its “Vision 2021,” Iis petroleum- and natural-gas-reliant economy is committed to sustainable development in order to emerge as the Gulf Cooperation Council’s, or GCC’s, most diversified economy. This includes the digitization of the economy, which has become a priority during the COVID-19 pandemic.
Related: Not like before: Digital currencies debut amid COVID-19
A green economy for sustainable digitization
The first virtual Abu Dhabi Sustainability Week Summit 2021 was broadcast live around the world in English and Arabic on YouTube, receiving over 100,000 views during the event from participants hailing from over 175 countries, and it featured over 500 influential global leaders from government, business and technology who explored the social, economic and technological opportunities supporting a sustainable green recovery from the pandemic.
At the summit, GCC leaders reconfirmed their decarbonization pledges “to save the equivalent of 354 million barrels of oil through the deployment” of renewable energies. This represents a 23% reduction in oil consumption to reduce the power sector’s carbon dioxide emissions by 22%, according to the latest figures from the International Renewable Energy Association.
In his opening address, Sultan Ahmed Al Jaber — the UAE’s minister of industry and advanced technology, special envoy for climate change, and chairman of clean energy company Masdar — pointed out that with the COVID-19 pandemic, society is now witnessing the implementation of artificial intelligence, machine learning and the digitization of different spheres of life all over the world. Accordingly, electrification, decarbonization and digitization initiatives have become increasingly important across all industries.
Masdar’s solar energy initiatives
New digital technologies require a high consumption of electricity, which in the UAE is currently produced predominantly using fossil fuels that adversely impact the environment. Given the UAE’s vast hydrocarbon resources, Masdar is aiming to become a major blue hydrogen producer and contribute to the nation’s efforts to cut polluting carbon emissions by nearly a quarter. Masdar recently reached an agreement with Abu Dhabi’s Department of Energy and five additional institutions to develop clean hydrogen fuel solutions.
But the UAE’s Paris Agreement commitment to zero emissions by 2050 is heavily reliant on solar energy to diversify Abu Dhabi’s energy sector into renewable sources. Solar energy is seen as an anchor to Masdar’s renewable strategies from many perspectives. In Abu Dhabi, it is building the world’s largest solar power plant, as deserts are some of the best places to harvest solar power. They are never short of sunlight and are rich in silicon — the raw material for the semiconductors from which solar cells are made. Another benefit to installing solar panels in the desert, according to a 2018 study, is that it may create a more humid environment that causes vegetation to spread to combat desertification.
Masdar City: The UAE’s aerospace and green technology zone
Developed by Masdar, Abu Dhabi’s Masdar City is one of the world’s most sustainable urban communities, offering a strategic base through which companies can build their networks locally and globally and can explore multiple investment opportunities and test innovative new technologies from inception through to implementation to help the UAE diversify its economy.
Housing a free zone area, the city has more than 900 organizations, from international conglomerates to startups, developing innovative technologies in the areas of energy, water efficiency, mobility, space, blockchain technology and artificial intelligence to address the world’s most critical sustainability challenges in more than 30 countries.
UAE Space Agency
Based in Masdar City, the UAE Space Agency contributes to supporting a sustainable national economy by developing satellites used in natural resource mapping, environmental monitoring, land-use planning and security, and it has also launched a probe to Mars.
The UAE government has made the digitization of its economy a priority in order to bring efficiency to government, creativity to industry, and build international leadership. To accomplish this goal, the UAE has established in Masdar City the world’s first graduate-level, research-based artificial intelligence university, Mohamed bin Zayed University of Artificial Intelligence, which welcomed its first students in January 2021.
The UAE also adopted the Emirates Blockchain Strategy 2021 and The Dubai Blockchain Strategy, which have undertaken several blockchain projects. SustVest is a crowd-investing blockchain-based platform that lets people invest in solar projects and earn returns from consumers who use their funding to install solar panels. The company is based in the Dubai Silicon Oasis Authority and has built its solution on the Nem blockchain. Its founder, Hardik Bhatia, explained:
“The global rooftop solar segment is booming with opportunities, and is valued at over $66 billion. Emerging economies are looking to transition to solar as it offers a green and cheap alternative to conventional energy sources. SustVest enables this transition in emerging economies by crowdfunding rooftop solar projects in emerging economies on its platform. We tokenize solar projects granular to the level of individual solar cells, and investors purchasing these tokens can earn dividends generated by the sale of electricity from these individual solar cells. We are opening the gates for retail investment into solar space, and we do so by tokenizing the projects to reduce the barrier of entry and creating a secondary marketplace for providing liquidity to investors.”
The Central Bank of the United Arab Emirates, along with the Saudi Central Bank, is developing a state-backed bilateral central bank digital currency, “Aber.” Aber is initially set to help the UAE and Saudi Arabia make more cost-effective bank-to-bank, cross-border payments and financial settlements using blockchain technology on a probationary basis, and according to official statements, it will be exclusively available to a limited number of banks. Eventually, Aber will be used globally on China’s blockchain-based service network, or BSN, which will support future CBDCs from various countries such as the UAE.
Related: The United Arab Emirates chase crypto and blockchain adoption
Crypto-asset regulations in the UAE
The UAE prioritizes blockchain and distributed ledger technology and has launched various related ventures, especially since the COVID-19 pandemic. Nevertheless, cryptocurrency regulation in the nation remains limited.
Toward the end of 2020, the UAE’s Securities and Commodities Authority, or SCA, published “The Authority’s Chairman of the Board of Directors Decision No. (21/R.M) of 2020 Concerning the Regulation of Crypto Assets.” The SCA’s decision lays out its licensing regime for anyone who wishes to offer crypto assets within the UAE, including exchanges, crowdfunding platforms, initial coin offerings, custodians, and other services that use crypto assets.
The Financial Services Regulatory Authority, or FSRA, of the Abu Dhabi Global Market considers crypto assets to have characteristics like those of shares, meaning they are to be treated as securities and are subject to information disclosure requirements related to risk and transactions. On the other hand, utility tokens and non-fiat cryptocurrencies are considered commodities and are not subject to market regulations. Law No. 20 of 2018 on Anti-Money Laundering defines laundered funds to be assets in whatever form, including digital currencies. Article 3 of Law No. 8 of 2017 on value-added tax imposes a 5% tax on imported and exported commodities. This tax may apply to utility tokens and non-fiat cryptocurrencies, as the FSRA considers them to be commodities.
The UAE does not have a signed tax treaty agreement with the United States. However, according to the Conduct of Business Rulebook, crypto-asset businesses are obligated to declare international income for tax purposes according to the requirements of the intergovernmental Foreign Account Tax Compliance Act agreement between the UAE and the United States.
A green recovery is an absolute imperative for a sustainable social and economic future in the post-pandemic world, as pointed out by Alok Sharma, president of the 26th United Nations Climate Change Conference of the Parties — better known as COP26 — who praised Masdar’s undertakings in developing green energy technologies.
Related: The need to report carbon emissions amid the coronavirus pandemic
Finding financing for this green transition will likely not be too challenging, according to Khaldoon Khalifa Al Mubarak, managing director and group CEO of Mubadala Investment Corporation. Because a tectonic paradigm shift has occurred since the COVID-19 pandemic, with the markets pricing climate risk into the value of securities, there is a fundamental reallocation of capital toward sustainable investing to ensure a green recovery in a post-COVID-19 world. As Laurence Fink, chairman and CEO of BlackRock — the world’s largest asset manager — pointed out:
“I believe that the pandemic has presented such an existential crisis — such a stark reminder of our fragility — that it has driven us to confront the global threat of climate change more forcefully and to consider how, like the pandemic, it will alter our lives. It has reminded us how the biggest crises, whether medical or environmental, demand a global and ambitious response.”
The views, thoughts and opinions expressed here are the author’s alone and do not necessarily reflect or represent the views and opinions of Cointelegraph.
Selva Ozelli, Esq., CPA, is an international tax attorney and certified public accountant who frequently writes about tax, legal and accounting issues for Tax Notes, Bloomberg BNA, other publications and the OECD.
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text:
Are you struggling with credit debt? Do you owe more than what you're making? Are overdrafts growing to be a massive concern? Are you fed up with viewing delinquent bills? Contact a great debt negotiation agency in Industry, Pennsylvania and end your struggles!
Do-it-Yourself Debt Settlement And Information About Debt Negotiation
With debt negotiation, you contact each of your collectors to create an arrangement in which you can pay them an amount that is less than your balance to settle your account. Often, you'll be able to seek the services of a service that will do that for you. You're able to attain settlement by setting up an agreement in which the creditor accepts a more affordable payment than what is owed. If you're unable to pay back the whole amount and are far past due on payments, you are likely to have a reduced total. Financial distress and difficulties will let you receive settlement opportunities.
Debt negotiation is worth looking at before thinking of a bankruptcy proceeding. You'll be able to avoid an entire life of damage by staying away from going bankrupt. Since collectors could end up having nothing at all, they like to negotiate for a settlement. You'll be able to fully overcome the debt in this way. The account is going to be made good for lower than your balance. There will be no additional endeavors to recover on the financial debt.
You are able to negotiate any type of financial debt. Whether it's medical bills, credit cards, or student loans, or another sort of debt, you are able to settle a repayment plan or decreased sum so that they get something and you have your financial debt repaid. It is a bit more challenging to discuss debt negotiation for a secured loan, such as where your vehicle or house was offered as collateral, unless of course you're ready to give up your car or home. A lot of people are even able to negotiate with the IRS. It is a little less common to settle education loan debt.
Negotiation will likely influence your credit standing. This is a thing that all the credit reporting agencies knows about. You'll be able to see the information from your settlement on your credit profile.
You don't have to do this on your own. There is also an option of employing a debt settlement organization. If you are unfamiliar with the procedure of negotiation or would like to avoid the phone calls, using a debt negotiation agency is a good option. It might be frustrating, annoying, and a little frightening to negotiate the debt by yourself. The very idea of this process can inspire fear and anxiety. And then, there are people who simply haven't got enough time for it. If you do not want to do it by yourself for any reason at all, think about working with a debt negotiation organization. If you're the type who likes to handle these matters on your own, you are not the only one. Some people are much more personal or want to steer clear of having a "middle man." Remember to research your options before you decide to retain the services of a debt negotiation service. This can be the most effective way to avoid services with lots of complaints filed against them and the ones that want upfront costs. If you opt for a respected organization, you should be fine.
Get in touch with creditors whenever you experience financial hardship. You should speak personally with the collectors and creditors if you can. Always keep a record of every conversation, call, email, or letter between you and the creditors. If you are not confident with this process or don't have time to devote, it is advisable to do business with a debt settlement company.
Always ask for a written copy of the arrangements, and you'll want to examine them diligently. Each settlement needs to be supported by a written arrangement. Save this info for your records. If a disagreement occurs, you should have them. You may even require them once you file taxes.
Be Sure That Do it Yourself Debt Settlement Is The Best Solution For Your Situation
Many people have reported success with do-it-yourself debt settlement. You'll be able to begin by simply contacting customer service with each credit card company. Having said that, more often than not, a credit card provider will only work with a consumer who is way past due on his payments and who wants to make one lump sum payment. Repayment plans aren't an option. The credit card company will need you to produce a solitary one time payment.
Do-it-yourself debt settlement saves funds that would be given to a debt settlement service. You should also have more command over this process.
What Are The Advantages From A Professional Debt Settlement Program?
It's usually far better to make use of a qualified professional debt negotiation company. They'll make wonderful deals because of their lengthy relationships with credit card companies. On your own, it's not that simple to get this kind of good deal. Plus, they can arrange all of your monthly debt repayments to be routed through a sole payment per month which goes through them. This can make things simple.
Doing the work on your own will often be less helpful than doing the work with the help of a good debt settlement service. Debt negotiation organizations will take a portion of the savings of the debt to pay for their expert services. Through the negotiation company, up to fifty percent of the current balances could be packed into a bigger bulk settlement, and that is a far better agreement. The established associations with creditors lets them come up with a far better rate. With the overall economy right now, more and more credit card companies may be prepared to negotiate their credit debt rather than adding to their already big written off debt.
The Drawbacks of The Debt Settlement Method
Damages credit history: FICO scores can decline with a debt settlement. Yet, if you're able to acquire a paid in full document from the collector, the credit report of the debtor won't exhibit any sign of a debt negotiation. The credit score improves again as accounts are settled. You'll find opportunities with most debt negotiation agencies to help you to boost your credit.
Possible getting sued: Any time anyone doesn't pay off a debt, they run the risk of lawsuit. Within the debt negotiation process, the account of the person in debt will remain in default. Whenever debts are in default, lawsuits could be an issue. A lump sum payment is typically the only method to settle debt for less than what's due.
Ineligible financial debt The results of your discussions can be affected by the sorts of personal debt you've got. Some kinds of debt are completely unaffected by debt settlement. For instance, you can't count on seeing any relief from domestic judgments, tax liens, or student loan debt. There are also the creditors that don't even want to think about negotiation.
IRS taxation considerations: Because debt settlementss are claimed as taxed income, many choose to avoid the approach. But, the IRS does not make people claim the given debt if the taxpayer was at an insolvent condition at the time when the creditor forgave debt.
Finding The Right Industry, Pennsylvania Debt Settlement Organization
Will the service cost you anything prior to lowering your debt?
You must think about this before choosing what debt settlement agency you intend to deal with. A respectable service will not impose a substantial fee to begin concentrating on your debt predicament. A small fee, or something like an application cost, is to be expected. Make sure you aren't asked to pay more than this this up front.
Are there difficulties with customer care? How many? What is their Better Business Bureau status look like?
Search the web to know more about the business and what others think about it. It's not hard to observe how past customers feel about a service through reviews. It is also useful to see whether complaints are filed by checking with the State Attorney General and the chamber of commerce in your town.
Did you check for connection to the American Fair Credit Council?
The advancement of good methods in the debt negotiation industry is the objective of the American Fair Credit Council.It is essential to shield the public from unfair methods by debt negotiation agencies, and the AFCC focuses on this objective. Association with the AFCC demands sticking to a strict range of regulations including adequate disclosure for customers along with the promotion of practices that strengthen the client satisfaction and completion .
Did the organization clarify how their process operates?
You need to receive all the facts about the whole process of debt negotiation and exactly how it works. The discussion should include every option you have, such as credit counseling, credit and debt consolidation, and bankruptcy. If they don't go over each option with you, be on your guard. You shouldn't ever feel like the sale is a bigger factor than your own interests.
Can you get web access to your account to view and monitor your financial progression?
Transparency is crucial when making use of a debt settlement service. The client needs to understand what is being accomplished on their behalf and also have easy access to account status. In a lot of situations, debt negotiation companies are not that large and are not going to possess the resources to supply their customers with this kind of details. You want to work with one that will achieve this.
For you to view negotiation offers, contact customer service, access accounts, and monitor advancement, you have got to work with the best agency.
Now, you know what to search for in a superior debt negotiation organization. This is the way you can steer clear of the programs with inadequate reputations, ability, and service.
If you've not had a chance to work together with a professional debt settlement agency, and you have just tried it on your own before, then this should come as a huge help for you. Bear in mind, you will not be required to pay much up-front, and you'll be able to get your debt dealt with at last.

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MBB: South Carolina upends No. 25 Michigan
South Carolina beat No. 25 Michigan 61-46 Wednesday evening. Sindarius Thornwell was spectacular, leading all players with 21 points, 10 rebounds, and three blocks.
The game was a defensive and officiating struggle. Both teams had a poor night shooting, and near constant foul calls prevented either team from getting into any sort of rhythm. But South Carolina worked through the circumstances, and Michigan did not.
Frank Martin was thrilled with how his players adjusted and committed to the game plan. On Tuesday assistant coach Matt Figger had the players identify the keys to the game and wrote them down.
"We had 10 keys, five defensive and five offensive on the board, and those guys did exactly what they said they needed to do to win the game," Martin said.
In the second half, the Gamecocks attacked the rim relentlessly, either with post-ups or penetration. On the other hand, the Wolverines continued to fire away from long range, even as they shot themselves out of the game.
"Their defense really affected us," Michigan coach John Beilein said. "They rebounded the ball very well. We got beat by a better team."
Michigan missed 24 of 26 three point attempts and finished the game with just two assists on ten made baskets. Michigan shot 19 percent from the floor and an abysmal 14 percent in the second half. Players missed some open looks, but South Carolina did an outstanding job of contesting every shot.
"I don't think we were that open," Beilein said. "It wasn't like they were wide open. Their defense was good."
Michigan scored just eight points in the paint and not a single fast break point. Meanwhile South Carolina scored 36 points in the paint and had eight fast break points. South Carolina held a 40-36 rebounding edge and forced 16 turnovers.
"Our biggest thing to focus on was guarding the ball and don't let them make passes," Thornwell said. "Stay in line and disrupt their offense."
The game came down to the Gamecocks' ability to get to the rim and make shots. Forwards Maik Kotsar and Chris Silva both scored 10 points, but it was Thornwell who made play after play to pull away from the Wolverines. He defended every position, rebounded over much taller players, and got to the rim at will. Thornwell has had an outstanding start to his senior season and is in the best shape of his career. He is also playing without any pain in his knees, something that has hampered him in the past.
" I couldn't really go by people and couldn't really get under my shot," Thornwell said. "Now with my body I can go by people and make my shots. It boosts my confidence a lot."
Notes: TeMarcus Blanton did not play due to an abdominal strain. ... Michigan was at least consistent with its assists. It had one assist in each half. ... Sedee Keita, who missed the first four games of the season with a wrist injury, saw his first action against Michigan. He did not score and had only one rebound in eight minutes. However, he drew praise from Martin for his play. ... Someone's phone went off while Martin was speaking after the game. "Get on the line!" he yelled. Then he said, "It's okay. I'm in a good mood today." ... Duane Notice chipped in 13 points for South Carolina. ... South Carolina will play at Michigan either next season or the season after in the return game. ... South Carolina returns to action Saturday against Syracuse in Brooklyn.