Responsiveness to therapy for liver disorders

Liver disorders, such as hepatitis, are major public health concerns. For example, hepatitis C is estimated to affect 200 million people worldwide. Patients with liver damage resulting from hepatitis C may develop chronic liver diseases, such as cirrhosis and hepatocellular carcinoma. Hepatitis C can be treated with interferon α and ribavirin. However, interferon α or ribavirin therapy causes significant side effects and is expensive. More importantly, only about 50% hepatitis C patients are responsive to the treatment. New therapies have been vigorously sought. Although several drug candidates are now being evaluated, the progress is rather slow due to a lack of appropriate systems for determining a patient's response to new therapies. Thus, there is a need for a reliable system and method for predicting a patient's response to treatment of hepatitis C and other liver disorders.

A patient's response to viral therapy is associated with various viral factors, e.g., the viral level, viral genotype, and mutation in certain viral proteins, and host factors, e.g., the patient's age, gender, race, host immune response, HLA alleles, and other genetic compositions, such as polymorphisms.

Single nucleotide polymorphisms (SNPs), a set of single nucleotide variants at genomic loci, are distributed throughout a genome. An SNP can be “allelic.” More specifically, due to polymorphism, some members of a species have the unmutated sequence (i.e., the wild-type allele) and others have a mutated sequence (i.e., the mutant allele). In humans, a polymorphism or a set of polymorphisms may be associated with a genetic disorder. In addition, patients having different SNP genotypes respond to the same treatment differently. Therefore, an SNP genotype of a patient is expected to provide individualized guidance for preventing and treating various human disorders.

This invention relates to sets of target nucleic acids, primers, and methods that can be used to predict the responsiveness of a subject to a therapy for treating hepatitis C and other liver disorders. The target nucleic acids contain one or more SNPs. The primers can be used in polymerase chain reaction (PCR) amplification for obtaining sequences containing the SNPs or can be used in single-base-extension for SNP typing.

Accordingly, one aspect of this invention features a set of SNP-containing target nucleic acids that include one or more of (i) a first target nucleic acid obtained from amplification of the human Adenosine Deaminase-RNA-specific (ADAR) gene nucleic acid template with a first pair of primers, each containing an oligo-nucleotide selected from the ADAR gene region; (ii) a second target nucleic acid obtained from amplification of the human Caspase 5 (CASP5) gene nucleic acid template with a second pair of primers, each containing an oligo-nucleotide selected from the CASP5 gene region; (iii) a third target nucleic acid obtained from amplification of the human Fibroblast Growth Factor 1 (FGFL) gene nucleic acid template with a third pair of primers, each containing an oligo-nucleotide selected from the FGF1 gene region; (iv) a fourth target nucleic acid obtained from amplification of the human Interferon Consensus Sequence Binding Protein 1 (ICSBP1) gene nucleic acid template with a fourth pair of primers, each containing an oligo-nucleotide selected from the ICSBP1 gene region; (v) a fifth target nucleic acid obtained from amplification of the human Interferon-Induced Protein 44 (IFI44) gene nucleic acid template with a fifth pair of primers, each containing an oligo-nucleotide selected from the IFI44 gene region; (vi) a sixth target nucleic acid obtained from amplification of the human Phosphoinositide-3-Kinase Catalytic Gamma Polypeptide (PIK3CG) gene nucleic acid template with a sixth pair of primers, each containing an oligo-nucleotide selected from the PIK3CG gene region; (vii) a seventh target nucleic acid obtained from amplification of the human Transporter 2 ATP-Binding Cassette Sub-Family B (TAP2) gene nucleic acid template with a seventh pair of primers, each containing an oligo-nucleotide selected from the TAP2 gene region; and (viii) an eighth target nucleic acid obtained from amplification of the human Transforming Growth Factor, Beta Receptor Associated Protein 1 (TGFBRAP1) gene nucleic acid template with a eighth pair of primers, each containing an oligo-nucleotide selected from the TGFBRAP1 gene region.

A “target nucleic acid” refers to an isolated nucleic acid that contains one or more SNPs of interest. An isolated nucleic acid refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA and (b) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.

Each of the above-mentioned target nucleic acids has a nucleotide at a single nucleotide polymorphism site and is 20-1,000 (e.g., 200-1,000) nucleotides in length.

Each of the above-mentioned oligo-nucleotide is 10-50, such as 14-50 (e.g., 14-40), nucleotides in length. A primer containing such oligo-nucleotide is 10-60 (e.g., 12-55) nucleotides in length. Exemplary sequences of the target nucleic acids (SEQ ID NOs: 1-74) are listed in Table 1 below. Exemplary primers (SEQ ID NOs: 75-230), including forward (“F”) and reverse (“R”) primers, are listed in Table 2 below.

In one embodiment of the set of target nucleic acids of this invention, (i) the first target nucleic acid contains SEQ ID NO: 1, 2, 3, 4, 5, or 6; (ii) the second target nucleic acid contains SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13; (iii) the third target nucleic acid contains SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, or 22; (iv) the fourth target nucleic acid contains SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, or 30; (v) the fifth target nucleic acid contains SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; (vi) the sixth target nucleic acid contains SEQ ID NO: 41, 42, 43, or 44; (vii) the seventh target nucleic acid contains SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, or 52; and (viii) the eighth first target nucleic acid contains SEQ ID NO: 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74. For the oligo-nucleotides, (i) the oligo-nucleotides in the first pair of primers are, SEQ ID NOs: 75 and 76, respectively; SEQ ID NOs: 77 and 78, respectively; or SEQ ID NOs: 79 and 80, respectively; (ii) the oligo-nucleotides in the second pair of primers are, SEQ ID NOs: 81 and 82, respectively; SEQ ID NOs: 81 and 177, respectively; SEQ ID NOs: 83 and 84, respectively; SEQ ID NOs: 83 and 178, respectively; SEQ ID NOs: 85 and 86, respectively; SEQ ID NOs: 87 and 88, respectively; SEQ ID NOs: 89 and 90, respectively; SEQ ID NOs: 89 and 181, respectively; SEQ ID NOs: 91 and 92, respectively; SEQ ID NOs: 93 and 94, respectively; SEQ ID NOs: 179 and 180, respectively; SEQ ID NOs: 182 and 183, respectively; or SEQ ID NOs: 184 and 185, respectively; (iii) the oligo-nucleotides in the third pair of primers are, SEQ ID NOs: 95 and 96, respectively; (iv) the oligo-nucleotides in the fourth pair of primers are, SEQ ID NOs: 97 and 98, respectively; SEQ ID NOs: 186 and 98, respectively; SEQ ID NOs: 99 and 100, respectively; SEQ ID NOs: 99 and 187, respectively; SEQ ID NOs: 101 and 102, respectively; SEQ ID NOs: 101 and 188, respectively; SEQ ID NOs: 103 and 104, respectively; SEQ ID NOs: 103 and 189, respectively; SEQ ID NOs: 105 and 106, respectively; SEQ ID NOs: 105 and 190, respectively; SEQ ID NOs: 107 and 108, respectively; SEQ ID NOs: 191 and 108, respectively; SEQ ID NOs: 109 and 110, respectively; or SEQ ID NOs: 192 and 110, respectively; (v) the oligo-nucleotides in the fifth pair of primers are, SEQ ID NOs: 111 and 112, respectively; SEQ ID NOs: 113 and 114, respectively; SEQ ID NOs: 115 and 116, respectively; SEQ ID NOs: 115 and 195, respectively; SEQ ID NOs: 117 and 118, respectively; SEQ ID NOs: 119 and 120, respectively; SEQ ID NOs: 121 and 122, respectively; SEQ ID NOs: 123 and 124, respectively; or SEQ ID NOs: 193 and 194 respectively; (vi) the oligo-nucleotides in the sixth pair of primers are, SEQ ID NOs: 125 and 126, respectively; SEQ ID NOs: 125 and 196, respectively; SEQ ID NOs: 127 and 128, respectively; SEQ ID NOs: 197 and 128, respectively; SEQ ID NOs: 129 and 130, respectively; SEQ ID NOs: 129 and 198, respectively; SEQ ID NOs: 131 and 132, respectively; or SEQ ID NOs: 199 and 200, respectively; (vii) the oligo-nucleotides in the seventh pair of primers are, SEQ ID NOs: 133 and 134, SEQ ID NOs: 201 and 134, respectively; SEQ ID NOs: 135 and 136, respectively; SEQ ID NOs: 202 and 136, respectively; SEQ ID NOs: 137 and 138, respectively; SEQ ID NOs: 203 and 138, respectively; SEQ ID NOs: 139 and 140, respectively; SEQ ID NOs: 204 and 140, respectively; SEQ ID NOs: 141 and 142, respectively; SEQ ID NOs: 205 and 142, respectively; SEQ ID NOs: 143 and 144, respectively; SEQ ID NOs: 206 and 144, respectively; SEQ ID NOs: 145 and 146, respectively; SEQ ID NOs: 145 and 207, respectively; SEQ ID NOs: 147 and 148, respectively; or SEQ ID NOs: 208 and 148, respectively; and (viii) the oligo-nucleotides in the eighth pair of primers are, SEQ ID NOs: 149 and 150, respectively; SEQ ID NOs: 151 and 152, respectively; SEQ ID NOs: 153 and 154, respectively; SEQ ID NOs: 155 and 156, respectively; SEQ ID NOs: 157 and 158, respectively; SEQ ID NOs: 217 and 158, respectively; SEQ ID NOs: 159 and 160, respectively; SEQ ID NOs: 159 and 218, respectively; SEQ ID NOs: 161 and 162, respectively; SEQ ID NOs: 163 and 164, respectively; SEQ ID NOs:163 and 227, respectively; SEQ ID NOs: 165 and 166, respectively; SEQ ID NOs: 167 and 168, respectively; SEQ ID NOs: 169 and 170, respectively; SEQ ID NOs: 171 and 172, respectively; SEQ ID NOs: 173 and 174, respectively; SEQ ID NOs: 175 and 176, respectively; SEQ ID NOs: 209 and 210, respectively; SEQ ID NOs: 211 and 212, respectively; SEQ ID NOs: 213 and 214, respectively; SEQ ID NOs: 215 and 216, respectively; SEQ ID NOs: 219 and 220, respectively; SEQ ID NOs: 221 and 222, respectively; SEQ ID NOs: 223 and 224, respectively; SEQ ID NOs: 225 and 226, respectively; SEQ ID NOs: 228 and 229 respectively; or SEQ ID NOs: 230 and 176, respectively.

In an preferred embodiment of the set of target nucleic acids of this invention, the first target nucleic acid contains SEQ ID NO: 1, 2, or 3; (b) the second target nucleic acid contains SEQ ID NO: 7, 8, 9, or 10; (c) the third target nucleic acid contains SEQ ID NO: 14 or 15; (d) the fourth target nucleic acid contains SEQ ID NO: 23, 24, 25, 26, 27, or 28; (e) the fifth target nucleic acid contains SEQ ID NO: 31, 32, 33, or 34; (f) the sixth target nucleic acid contains SEQ ID NO: 41, 42, or 43; (g) the seventh target nucleic acid contains SEQ ID NO: 45, 46, or 47; and (h) the eighth target nucleic acid contains SEQ ID NO: 53, 55, 56, or 57. For the oligo-nucleotides, (a) the oligo-nucleotides in the second pair of primers are, respectively, SEQ ID NOs: 81 and 82, SEQ ID NOs: 81 and 177, SEQ ID NOs: 83 and 84, SEQ ID NOs: 83 and 178, SEQ ID NOs: 85 and 86, SEQ ID NOs: 87 and 88, or SEQ ID NOs: 179 and 180; (b) the oligo-nucleotides in the fourth pair of primers are, respectively, SEQ ID NOs: 97 and 98, SEQ ID NOs: 186 and 98, SEQ ID NOs: 99 and 100, SEQ ID NOs: 99 and 187, SEQ ID NOs: 101 and 102, SEQ ID NOs: 101 and 188, SEQ ID NOs: 103 and 104, SEQ ID NOs: 103 and 189, SEQ ID NOs: 105 and 106, or SEQ ID NOs: 105 and 190; (c) the oligo-nucleotides in the fifth pair of primers are, respectively, SEQ ID NOs: 111 and 112, SEQ ID NOs: 113 and 114, SEQ ID NOs: 115 and 116, SEQ ID NOs: 115 and 195, SEQ ID NOs: 117 and 118, or SEQ ID NOs: 193 and 194; (d) the oligo-nucleotides in the sixth pair of primers are, respectively, SEQ ID NOs: 125 and 126, SEQ ID NOs: 125 and 196, SEQ ID NOs: 127 and 128, SEQ ID NOs: 197 and 128, SEQ ID NOs: 129 and 130, or SEQ ID NOs: 129 and 198; (e) the oligo-nucleotides in the seventh pair of primers are, respectively, SEQ ID NOs: 133 and 134, SEQ ID NOs: 201 and 134, SEQ ID NOs: 135 and 136, SEQ ID NOs: 202 and 136, SEQ ID NOs: 137 and 138, or SEQ ID NOs: 203 and 138; and (f) the oligo-nucleotides in the eighth pair of primers are, respectively, SEQ ID NOs: 149 and 150, SEQ ID NOs: 151 and 152, SEQ ID NOs: 153 and 154, SEQ ID NOs: 209 and 210, SEQ ID NOs: 211 and 212, or SEQ ID NOs: 213 and 214.

Another aspect of this invention features a set of nucleic acids comprising one or more of the above-described first, second, third, fourth, fifth, sixth, seventh, and eighth pairs of primers.

A further aspect of this invention features a method of evaluating responsiveness of a subject to a drug. The method includes (1) providing a nucleic acid sample from a subject; (ii) determining a single nucleotide polymorphism genotype (SNP genotype) of a gene group that contains one or more of the human ADAR, CASP5, FGFI, ICSBP1, IFI44, PIK3CG, TAP2, and TGFBRAP1 genes; and (iii) comparing the single nucleotide polymorphism genotype with a predetermined SNP genotype. A subject is predicted to be responsive or non-responsive to the drug if the SNP genotype is identical to a predetermined SNP genotype.

A “genotype” refers to a specific allelic composition of an organism, a genome, or a part of a genome. In particular, this term refers to alleles of a particular gene or set of genes. A single nucleotide polymorphism or SNP genotype refers to the SNP composition of a gene or set of genes. It is presented as a string of nucleotides. For example, a region of human FGF1 gene on each chromosome contains two SNP sites (VGV567 and VGV679). If the two chromosomes of a subject have (1) C and T at the VGV567 sites and (2) A and A at the VGV567 sites, the FGF VGV567/VGV679 SNP genotype of this subject is represented as “CTAA.”

In one embodiment, the aforementioned nucleic acid sample contains one or more of the above-described SNP-containing target nucleic acids, which can be obtained from amplification of the corresponding human gene nucleic acid template with a pair of primers. The method can be used to evaluate drug responsiveness of a subject that has or is suspected of having a liver disorder, such as hepatitis (e.g., hepatitis C), liver fibrosis, or liver cirrhosis. Examples of a drug include type I interferon (e.g., interferon-alpha and interferon-beta), ribavirin, or their combination. In one example, a subject is predicted to be responsive or non-responsive to type I interferon if his or her SNP genotype is identical to a predetermined SNP genotype such as one of those reference genotypes listed in Table 4 in Example 1 below. A reference genotype refers to a SNP genotype known to be associated with the responsiveness or non-responsiveness to a certain drug. It can be determined in the manner described in Example 1 below.

The details of one or more embodiments of the invention are set forth in the accompanying description below. Other advantages, features, and objects of the invention will be apparent from the detailed description and the claims.

This invention relates to nucleic acids and a method of using the nuclei acids for predicting a subject's responsiveness to a drug based on an SNP genotype of the subject. The method requires determining a subject's SNP genotype of a gene or a set of genes and predicting the subject's responsiveness to the drug by comparing the SNP genotype with reference genotypes known to be associated with responsiveness or non-responsiveness to the drug.

One can select a gene or set of genes that encode proteins involved in the metabolism or signal transduction pathway of the drug of interest. For example, interferon (IFN) or ribavirin is often used in treating hepatitis C. Genes encoding proteins involved in IFN signaling pathways or immunomodulation can be selected for SNP genotyping. As demonstrated in Example 1 below, the SNP genotypes of a set of certain genes showed statistically significant differences between subjects responsive to IFN/ribavirin and subjects non-responsive to IFN/ribavirin. To predict a subject's responsiveness to IFN/ribavirin, one can determine the subject's corresponding SNP genotype of one or more of these genes.

Many technologies known in the art can be used to genotype SNPs (see, e.g., Kwok, Pharmacogenomics, 2000, vol 1, pp 95-100. “High-throughput genotyping assay approaches”). These technologies are based on direct sequencing, allele specific oligonucleotide hybridization, oligonucleotide elongation by dideoxynucleotides or ddNTPs optionally in the presence of deoxynucleotides, ligation of allele specific oligonucleotides, or cleavage of allele specific oligonucleotides. Each of these technologies can be coupled to a detection system such as measurement of direct or polarized fluorescence, or mass spectrometry.

The genotyping assays can be carried out on a product obtained from amplification of the DNA of a subject. This product and corresponding primers are selected to cover a polynucleotide region containing a SNP of interest. Amplification techniques known in the art include, but are not limited to, cloning, polymerase chain reaction (PCR), polymerase chain reaction of specific alleles (ASA), ligase chain reaction (LCR), nested polymerase chain reaction, self sustained sequence replication (Guatelli, et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), and Q-Beta Replicase (Lizardi, et al., 1988, Bio/Technology 6:1197).

Amplification products can be assayed in a variety of ways, including size analysis, restriction digestion followed by size analysis, detecting specific tagged oligonucleotide primers in the reaction products, allele-specific oligonucleotide (ASO) hybridization, allele specific 5′ exonuclease detection, primer extension, sequencing, hybridization, and the like. PCR based amplification techniques include multiplex amplification of a plurality of markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, it is possible to amplify different markers with primers that are differentially labeled and thus can each be differentially detected. Of course, hybridization based detection techniques, e.g., microarraies, allow the differential detection of multiple PCR products in a sample simultaneously.

After determining a subject's SNP genotype, one can compare it with a reference genotype known to be associated with responsiveness or non-responsiveness to a drug. If the SNP genotype is identical to a reference genotype associated with responsiveness (or non-responsiveness), the subject is predicted to be responsive (or non-responsive). When comparing with reference SNP genotypes of more than one gene, one might obtain contradictory conclusions. In this case, one should take into account of the weight, i.e., prediction power, of each gene. Table 4 in Example 1 below lists exemplary reference SNP genotypes of 8 genes and corresponding weights.

For high throughput comparison, one can incorporate reference SNP genotypes of all genes of interest and the corresponding weights into a logical formula, which has a number of variables corresponding to the genes, respectively. After substituting a subject's SNP genotype of each gene for each variable, one can obtain a predicting result returned from the formula. An exemplary logical formula is shown below:

In the formula, “p” stands for the probability of a subject to be responsive to a treatment. “Gene” is a value (e.g., +1, −1, or 0) for a reference SNP genotype of a gene that is associated with the responsiveness or non-responsiveness to a drug. “Weight” represents” the weight/prediction power of each gene as mentioned above.

One can obtain reference SNP genotypes in a manner similar to that described in Example 1 below. More specifically, one can (1) select a sample group of subjects who are known to be responsive or non-responsive to a drug of interest, (2) SNP type a set of candidate genes, and (3) identify the degree of association between an SNP genotype and a responding status using statistic methods known in the art. The genotyping data is then analyzed to estimate the distribution frequency of the different alleles observed in the studied sample. Calculation of the allelic frequencies can be carried out with the assistance of a software program such as SAS® statistical package version 8, SAS-suite® (SAS) or SPLUS® (MathSoft). Comparison of the allelic distributions of a SNP across different ethnic groups can also be carried out by means of the software ARLEQUIN® and SAS-suite®.

The above-described methods and nucleic acids can be used to predict a hepatitis C patient's responsiveness to IFN. They can also be used in predicting IFN responsiveness of a subject that has or is suspected of having disorders that can be treated by IFN. Examples of these disorders include (1) diseases caused by viruses, such as hepatitis (A, B, C, D, E, F, G types) virus, HIV, influenza virus, herpes virus, adenovirus, human polyomavirus, human papilloma virus, human parbovirus, Mumps virus, human rotavirus, enterovirus, Japanese B Encephalitis virus, dengue virus, rubella virus, and HTLV; (2) diseases caused by bacteria, such as Staphylococcus aureus , hemolytic streptococcus , pathogenic Escherichia coli , enteritis vibrio, Helicobacter pylori, Campylobacter, Vibrio cholerae , dysentery bacilli, salmonellae, Yelsinia, Neisseria gonorrhoeae, Listeria, Leptospira, Legionella , spirochete, Mycoplasma pneumoniae , rickettsiae, chlamydiae, malaria plasmodia, dysentery amoeba, and pathogenic fungi; (3) diseases caused by parasites and Eumycetes, and (4) oncological diseases, such as retinoblastoma, Wilm's tumor, familial colonic polyposis, hereditary non polyposis colon cancer, neurofibromatosis, familial chest cancer, xeroderma pigmentosum, blain cancer, oral cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, thyroid cancer, mammary gland tumor, urinary tumor, virilia tumor, muliebria tumor, skin tumor, osteosarcoma, osteochondrosarcoma, leukemia, lymphoma, and solid tumor.

In addition, as shown in Example 2 below, SNPs of 8 genes were used to accurately predict patients' responsiveness to drugs. The results indicate that the 8 genes (i) are associated with the drug responsiveness and (ii) play important roles in the metabolism or signal transduction pathways of a drug, such as IFN or ribavirin. Accordingly, these genes and SNPs can be used as targets for drug development and as genetic markers for molecular diagnosis, respectively. Indeed, a compound that (i) binds to a protein encoded by one of the genes or (ii) regulates the expression level or activity of the protein is a drug candidate for treating one of the above-mentioned disorders, e.g., a viral infection, such as hepatitis C. Thus, within the scope of this invention is a method for identifying a compound or composition for treating one of the above-mentioned disorders, such as an infection with a virus, e.g., hepatitis virus. The method includes (1) obtaining a first polypeptide that contains a sequence encoded by one of the aforementioned 8 genes; (2) contacting a compound with the first polypeptide; and (3) detecting a binding between the first polypeptide and the compound. The compound is determined to be effective in treating the disorder if the compound binds to the first polypeptide, but not to a second polypeptide that is identical to the first polypeptide, except that the second polypeptide does not have the sequence encoded by one of the aforementioned 8 genes. Also within the scope of this invention is a method for identifying a compound for treating one of the above-mentioned disorders, e.g., a viral infection The method includes (1) obtaining a first system, e.g., a cell, containing or capable of expressing a polypeptide encoded by one of the 8 genes; (2) incubating the first system in a medium containing a compound; and (3) determining an expression or activity level of the polypeptide. The compound is determined to be effective in treating the disorder if the expression or activity level differs from that determined in the same manner from a second system except that the second system is incubated in a medium free of the compound.

The two specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.