Patent Application
20110185440
Although incorporated by reference in its entirety, no arguments or disclaimers made in the parent application apply to this divisional application. Any disclaimer that may have occurred during the prosecution of the above-referenced application(s) is hereby expressly rescinded. Consequently, the Patent Office is asked to review the new set of claims in view of the entire prior art of record and any search that the Office deems appropriate.
The present invention relates to a premature aging or Wolfram syndrome 2 (WFS2) animal model and use thereof.
Aging, or organismal senescence, is defined as gradual changes in an organism that “adversely affect its vitality and function, but most importantly, increases the mortality rate of an organism as a function of time”.
Aging can be characterized as the age-related decline of physiological functions necessary for the survival and reproduction of an organism. Common age-associated diseases connected to these functions include, but not limited to, arteriosclerosis, cancer, dementia and osteoporosis. To understand the primary causes of these diseases' onset and commencing of generalized malfunctions of multiple organ systems that potentially shorten life span and reduce fertility is central to understanding human aging.
A number of genetic components of aging have been identified using model organisms, ranging from the bakers' yeast (Saccharomyces cerevisiae), the soil roundworm (Caenorhabditis elegans), the fruit fly (Drosophila melanogaster), and the mouse (Mus musculus).
One approach to understanding the molecular basis of human aging is to find genes that determine inherited premature aging syndromes thereby causing rapid development of these senescence associated diseases early in life. To that end, mutant mice that display multiple phenotypes resembling accelerated aging have been developed in recent years. However, virtually all of them display partial spectrum of the senescence associated phenotypes.
CISD2 is the second member of the gene family containing the CDGSH iron sulfur domain. There are currently three members in this gene family: CISD1 (synonym ZCD1, mitoNEET), CISD2 (synonym ZCD2, Noxp70, Miner1) and CISD3 (synonym Miner2). CISD1 is an outer mitochondrial membrane protein that was originally identified as a target protein of the insulin sensitizer drug pioglitazone used to treat type 2 diabetes. CISD1 protein contains a transmembrane domain, a CDGSH domain and a conserved amino acid sequence for iron binding; biochemical experiments suggest that CISD1 is involved in the control of respiratory rates and regulates oxidative capacity. However, CISD2 and CISD3 are novel genes with previously uncharacterized functions. The only molecular documentation for CISD2 is that CISD2 was one of the markers for early neuronal differentiation in a cell culture study.
Recently CISD2 gene has been identified as the second causative gene associated with Wolfram syndrome (WFS; MIM 222300), which is an autosomal recessive neurodegenerative disorder. Wolfram syndrome is highly variable in its clinical manifestations, which include diabetes insipidus, diabetes mellitus, optic atrophy and deafness; thus, it is also known as the DIDMOAD syndrome. Positional cloning and mutation studies have revealed that WFS is a genetically heterogeneous disease with a complex molecular basis involving more than one causative gene in humans. A portion of WFS patients belonging to the Wolfram syndrome 1 group (WFS1; MIM 606201) carried loss-of-function mutations in the WFS1 (wolframin) gene, which encodes a transmembrane protein primarily localized in the endoplasmic reticulum (ER). In addition to this, a homozygous mutation of CISD2 gene has been identified in three consanguineous families with Wolfram syndrome and these patients have been classified as Wolfram Syndrome 2 (WFS2; MIM 604928). However, the function of the CISD2 protein in these patients and in all other organisms remains unknown and its physiological role has not been explored.
Significantly, CISD2 gene is located within the region on human chromosome 4q where a genetic component for human longevity has been mapped. Previously a research studied 137 sets of extremely old siblings (308 individuals in all) and conducted a genome-wide scan search for predisposing loci that might confer longevity; this linkage study revealed a single region on chromosome 4q and suggests that there may be at least one master gene contributing to lifespan control; however, the responsible gene has not been identified.
The present invention provides a Cisd2 knockout mouse with phenotype comprising mitochondrial breakdown and dysfunction, wherein Cisd2 is defined as SEQ ID NO. 1.
The present invention also provides a mouse model of Wolfram Syndrome 2 (WFS2) disease consisting of a Cisd2 knockout mouse.
The present invention further provides a method for screening a candidate agent for preventing or treating WFS2 disease comprising: (a) providing the mouse of claim 1; (b) adding said candidate agent into said mouse, and (c) determining the agent by identifying the desired therapeutic effects in ameliorating WFS2 disease associated phenotypes.
To adequately describe the present invention, references to embodiments thereof are illustrated in the appended drawings. These drawings herewith form a part of the specification. However, the appended drawings are not to be considered limiting in their scope.
The Cisd2-knockout mouse used in the present invention is equal to the Cisd2-knockout mouse in U.S. Application No. 60/849,089.
The term “Cisd2” as used herein means Mus musculus CDGSH iron sulfur domain 2, and the orthologous genes including Gret, ZCD2, Miner1, Noxp70, AI848398, 1500009M05Rik, 1500026J14Rik, 1500031D15Rik, and B630006A20Rik.
The present invention applies a mouse genetics approach and demonstrated that Cisd2 is involved in mammalian lifespan control and plays an essential role in mitochondrial integrity. Cisd2 deficiency causes mitochondria-mediated phenotypic defects in mice. Furthermore, cell culture and biochemical investigations revealed that Cisd2 is a mitochondrial protein. Additionally, Cisd2 knockout mice exhibit many clinical manifestations of WFS patients including early-onset degeneration of central (e.g. optic) and peripheral (e.g. sciatic) nerves and premature death, as well as impaired glucose tolerance. This study therefore provides an animal model for mechanistic understanding of WFS, specifically WFS2, pathogenesis.
The present invention recapitulates a more extensive set of early senescence associated features of human premature aging than those previously described. As such, the present invention provides an extremely useful model to elucidate premature aging or WFS2 disease in human.
Furthermore, the present invention offers an in vivo system to screen for agents in ameliorating the patho-physiological effects of premature aging or WFS2 disease.
A mutant animal of the present invention can be any non-human mammal, preferably a mouse. A mutant animal can also be, for example, any other non-human mammals, such as rat, rabbit, goat, pig, dog, cow, or a non-human primate. It is understood that mutant animals having a disrupted Cisd2 gene, as disclosed herein, or other mutant forms that eliminate the expression of Cisd2, can be used in methods of the invention. Thus, the mutant animal loss of all or a part of the Cisd2 gene function is due to a disruption of the Cisd2 gene
The present invention provides a line of genetically engineered mice either heterozygous (referred to as Cisd2+/−) or homozygous (referred to as Cisd24-) for the disrupted endogenous Cisd2 gene. This gene may be mutated by disrupting one or more of its exons by heterologous DNA sequences such as an HPRT cassette using standard molecular biological techniques. In addition, any mutant forms that eliminate the expression of Cisd2 can be used. The resulting Cisd2−/− mice exhibit a range of phenotypes similar to those of human aging including many physical or biochemical manifestations as detailed below. As such, these mice, Cisd2+/− and Cisd2−/− included, can be used as a model system to help delineate the molecular mechanisms underlying human premature aging or WFS2 disease.
The present invention also provides a cell or cell line from the Cisd2 knockout mouse, wherein the cell or cell line contains a targeted disruption in Cisd2 gene in which Cisd2 exon 3 has been disrupted. The cell or cell line is an undifferentiated cell which is selected from the group consisting of a stem cell, embryonic stem cell oocyte and embryonic cell.
The present invention further demonstrates a method of screening for agents useful in treating or preventing premature aging or WFS2 disease associated phenotypes or delaying the onset of premature aging consisting of administering candidate compounds to the Cisd2−/− mice or the cell or cell line derived from the Cisd2−/− and screening for the desired therapeutic effects.
The method for identifying a target gene having altered expression in a mutant Cisd2 mouse involves comparing the expression of one or more genes in a mutant mouse having a disrupted Cisd2 gene with the expression of said one or more genes in a wild type animal to identify a gene having altered expression in said mutant mouse, thereby identifying a target gene having altered expression in a mutant Cisd2 mouse.
As described in Example 7, Cisd2 mutant mice exhibited altered expression of genes in comparison to wild type mice in addition to other phenotypes described. For instance, Cisd2 knockout mice are characterized by decreased expression of BDNF (brain-derived neurotrophin factor). The altered expression of BDNF gene, as well as other genes having altered expression in a mutant Cisd2 mouse, indicates that Cisd2 normally regulates the expression of these genes in wild-type mice. Thus, these represent genes that can be modulated to reverse, or at least partially reverse, the physiological and biochemical characteristics of a Cisd2−/− phenotype. For example, restoring the expression of one of these Cisd2 regulated genes having altered expression in a mutant Cisd2 mouse to a level that can result in reversed phenotypes can be contemplated. Therefore, a compound that exhibits the said effect is a potentially useful therapeutic compound for treatment of premature aging associated phenotypes or possibly delaying the onset of premature aging.
As such, the present invention provides methods for identifying target genes having altered expression in a mutant Cisd2 mouse, as well as methods for identifying a compound that restores a target gene having altered expression in a mutant Cisd2 mouse to a level of expression achieving the desired therapeutic effect.
The methods of the invention for identifying a target gene having altered expression in a mutant Cisd2 mouse can involve comparing the expression of one or more genes contained within one or more organs of the mutant Cisd2 mice.
The method for identifying a compound that restores a target gene having altered expression in a mutant Cisd2 mouse to a therapeutic level of expression involves (a) contacting a target gene having altered expression in a mutant Cisd2 mouse with a test compound; (b) determining expression of said target gene, and (c) identifying a compound that modulates expression of said target gene to a level of expression consistent with a wild type level of expression.
The methods of the invention for screening for a compound that restores a target gene having altered expression in a mutant Cisd2 mouse to a more normal level of expression-involve contacting a sample exhibiting altered expression of a target gene characteristic of a mutant Cisd2 mouse with a test compound. A test compound can be any substance, molecule, compound, mixture of molecules or compounds, or any other composition which is suspected of being capable of restoring an expression level of a target gene to a more normal level.
Additionally, a test compound can be pre-selected based on a variety of criteria. For example, suitable test compounds having known modulating activity on a pathway suspected to be involved in a mutant Cisd2 phenotype can be selected for testing in the screening methods. Alternatively, the test compounds can be selected randomly and tested by the screening methods of the present invention.
A level of protein expression corresponding to a gene expression level also can be determined, if desired. A variety of methods well known in the art can be used to determine protein levels either directly or indirectly.
The methods of the invention for identifying a compound that restores a target gene having altered expression in a mutant Cisd2 mouse to a more normal level of expression can involve determining an activity of a target gene. The activity of a molecule can be determined using a variety of assays appropriate for the particular target. A detectable function of a target gene can be determined based on known or inferred characteristics of the target gene.
For use as a therapeutic agent, the compound can be formulated with a pharmaceutically acceptable carrier to produce a pharmaceutical composition, which can be administered to the individual, which can be a human or other mammal.
The methods of the invention can advantageously use cells isolated from a homozygous or heterozygous Cisd2 mutant mouse for a desired purpose. For example, these cells can be used as an in vitro method to screen agents for treating or preventing premature aging or WFS2 disease associated phenotypes or the onset of premature aging or the disease. In such a method, a compound is contacted with a cell having disrupted Cisd2 expression, and screen for modulation of the target gene as described above.
Thus, the invention provides methods of screening a large number of compounds using a cell-based assay, for example, using high throughput screening, as well as methods of further testing compounds as therapeutic agents in an animal model using the Cisd2 mutant mice.
The present invention is further directed to cell lines derived from the Cisd2+/−, or Cisd2−/− mice. These cell lines are useful in studying senescence at the cellular level and in drug screening assays. Cell lines derived from the brain, kidney, lung, stomach, intestine, spleen, heart, adipose, heart and liver tissues are especially useful in these applications.
In a preferred embodiment, the present invention is related to a Cisd2 knockout mouse with phenotype comprising mitochondrial breakdown and dysfunction, wherein Cisd2 is defined as SEQ ID NO. 1. In a more preferred embodiment, the mouse of the present invention has the phenotype comprises nerve demylination and neuron degeneration, cardiac and skeletal muscle degeneration, reduced body weight, prominent eyes and protruding ears, osteopenia, lordokyphosis, abnormal pulmonary function, opacity of the cornea, or skin atrophy and graying.
In a preferred embodiment, the Cisd2 gene is knockout by recombination with homologous nucleotide sequence. In a more preferred embodiment, knockout occurs in Cisd2 exon 3.
In a preferred embodiment, the mouse of the present invention has Cisd2 knockout steps comprising:
(a) an additional copy of a Cisd2 gene fragment consisting of a portion of intron 1, the entire exon 2, and a portion of exon 3 of the Cisd2 gene;
(b) a positive puromycin selection marker;
(c) a non-functional 3′-HPRT cassette; and
(d) a loxP site.
In a preferred embodiment, the present invention is related to a mouse model of Wolfram Syndrome 2 (WFS2) disease consisting of a Cisd2 knockout mouse aforementioned. In a more preferred embodiment, the present invention is related to a mouse model which is applied to screen a candidate agent for preventing or treating WFS2 disease.
The present invention also relates to a transgenic knock-out mouse whose genome comprises a homozygous disruption in its endogenous CDGSH iron sulfur domain 2 (CISD2) gene, wherein said CISD2 is defined as SEQ ID NO. 1, and wherein said homozygous disruption results in said transgenic knockout mouse exhibiting decreased levels of CISD2 as compared to a wild-type mouse, said mouse showing symptoms similar to the features of premature aging, said features consisting of optic atrophy, neurological features, ataxia, cardiac and skeletal muscle degeneration and nerve demyelination and neuron degeneration.
In a preferred embodiment, the optic atrophy of the transgenic knock-out mouse is a phenotype of progressive degeneration of optic nerve starts from age 3-week of the transgenic knock-out mouse. In a preferred embodiment, the ataxia of the transgenic knock-out mouse is a phenotype of progressive degeneration of sciatic nerve and unsteady gait starts from age 2-week of the mouse. In a preferred embodiment, the feature of cardiac and skeletal muscle degeneration in the transgenic knock-out mouse starts from age 3-week of the mouse. In another preferred embodiment, the feature of nerve demyelination and neuron degeneration in the transgenic knock-out mouse starts from age 3-week of the mouse.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
The mouse Cisd2 (SEQ ID NO. 1) was identified as the putative ortholog based on the remarkable protein sequence similarity (96% identity) to the human gene, Cisd2, located in the region where the longevity locus was previously mapped. It was then engineered and disrupted to understand its role in longevity in the present invention.
The expression pattern of the Cisd2 gene was characterized by examining the relative levels of mRNA present in adult mouse tissues (
The linearized targeting vector was electroporated into 129/SvEv embryonic stem (ES) cells. Selection medium containing puromycin and gancycloviour was applied 24 h after electroporation and maintained for 7 days. Resistant colonies were selected and re-seeded onto the feeder layer in a 96-well plate. DNA extracted from individual ES clone was isolated and detected by Southern blot analysis. The 3′ flanking probe used was a 1.7 kb BamHI-EcoRI fragment from exon3 (
The targeted ES cells were injected into C57BL/6 blastcysts and reimplanted into pseudopregnant female mice. Chimeric male mice were bred with C57BL/6 female. Genomic DNA was isolated from tail samples of the appropriate agouti progeny using proteinase K/SDS digestion and phenol/chloroform extraction method. Isolated DNA samples were further analyzed by Southern blot for germline transmission. The analysis confirmed the presence of both the endogenous and the disrupted alleles in the F1 heterozygotes. The heterozygous mice were intercrossed, and their offspring were genotyped.
Genotypes of offspring from heterozygous breeding demonstrated normal Mendelian ratios of homozygous (−/−), heterozygous (+/−) and wild-type (+/+). Fertility test of the Cisd2−/− males and females exhibited normal reproductive capability.
Southern blot analysis showed that the genomic DNA digested with EcoRI and hybridized with a probe shown in
Northern blot of total RNA prepared from the brain tissue of wild-type (+/+), heterozygous (+/−), and Cisd2-null (−/−) mice was probed with the 32P-labeled fragment identical to that used in Southern blot analysis. The probe detected a 2.8-kbp RNA band in samples from the wild-type and heterozygous but not from the homozygous animals. Hybridization of the same filter, after stripping of the Cisd2 probe, with a mouse glyceraldehyde-3-phosphate dehydrogenase (Gapd) probe confirmed that equal amounts of RNA were loaded on the gel.
Up to 3 weeks of age, Cisd2−/− mice appeared morphologically identical to their Cisd2+/+ littermates. However, starting around week 3, all of the Cisd2−/− mice started to display a wide range of senescence associated phenotypes shown in
Starting at 8-week old, the Cisd2−/− mice began to acquire a set of aged appearance remarkably similar to those displayed by patients with Hutchinson-Guilford progeria syndrome. These included prominent eyes and protruding ears (
Two anatomical characteristics commonly seen in aged human skins were reduced dermal thickness and subcutaneous adipose. Consistent with those features in human, the skin of 48-week-old Cisd2−/− mice exhibited phenotypes of massive hyperkeratosis, significant decrease of subcutaneous fat and muscle, and noticeably thickened dermis with expanded surface (
Tissue sections of the dorsal skin were stained with H&E and Masson's trichome staining. The thicknesses of the dermal, adipose and muscle layers were quantified by random measurements of the length of individual skin samples using SPOT Imaging Software Advance (DIAGNOSTIC Instruments Inc.).
Micro-computer tomography analysis detected a decrease of femur density in the 8-week-old Cisd2−/− mice compared with that of the age-match wild-type mice while the trabeculae of the femur in Cisd2−/− mice were noticeably thinner (
The bone samples of wild-type and Cisd2−/− mice were fixed in 10% buffered formalin phosphate, stored in 70% ethanol and examined by eXplore Locus SP Pre-Clinical Specimen MicroCT (GE Healthcare). Whole-body and femur scans were performed in the axial plane mounted in a cylindrical sample holder. The three-dimensional images of bones reconstructed from MicroCT scanning slices used to qualitatively evaluate bone structure and morphology. The quantitative data of bone tissue were separated from those for marrow and soft tissue and were analyzed by eXplore MicroView v. 2.0 Software Guide (GE Healthcare).
While showing no detectable skeletal abnormalities up to 8 weeks of age, radiographs of 12-week-old Cisd2−/− mice already displayed significant lordokyphosis (curvature of the spinal column) (
Indeed, the present invention observed decreases in various respiratory parameters as measured by plethysmography after 20-week old in the Cisd2−/− mice (
Respiratory parameters were measured in conscious mice with three genotypes by using plethysmography chambers where the mouse body was enclosed in a sealed chamber while the head was free. Thoracic movements were measured by pressure transducers that were linked to a Buxco amplifier system and respiratory parameters, then captured and analyzed by the Notocord HEM data acquisition system. Upon placement of the mice into the plethysmography chambers, tidal volume (TV) was determined 10 min at unrestrained condition. The formula for calculating Penh (Enhanced Pause) was: PEF/PIF×(Te/Rt-1), Where Te=Expiratory time, Rt=Relaxation time, PEF was Peak Expiratory Flow, and PIF was Peak Inspiratory Flow.
Muscle degeneration was detectable at 3-week old in the Cisd2−/− mice. There was a progressive degeneration of muscle fibres and the magnitude of the degeneration exacerbated with age (
To understand the basis for the morphological abnormality in the longitudinal fibers in Cisd2−/− mice, ultrastructure of muscle cells were examined by electron microscopy. Muscle tissues from wild-type and Cisd2−/− mice were fixed in a mixture of glutaraldehyde (1.5%) and paraformaldehyde (1.5%) in phosphate buffer at pH 7.3. These were postfixed in 1% OsO4, 1.5% potassium hexanoferrate, rinsed in cacodylate and 0.2 M sodium maleate buffers (pH 6.0), and block-stained with 1% uranyl acetate. Following dehydration, tissues were embedded in Epon and were ready for transmission electron microscopy. Degenerated myofilaments indicated by arrows (
Since myelin sheath degeneration was one of the clinical features in aging, the present invention sought to examine the state of peripheral nerves when Cisd2 gene expression was eliminated. In wild-type mice, the myelinated axons were enveloped with a myelin sheath formed by the fusion of many layers of plasma membrane from Schwann cells (
To investigate the effect of Cisd2 on transcription of other genes, select number of genes was examined. Reverse transcription was performed with 2 μg of total RNA and primed with random hexamers and Superscript III reverse transcriptase (Invitrogen Life Technologies). Real-time PCR was carried out on Roche LightCycler 480 Real-time PCR instrument, using TaqMan probe searched at Universal ProbeLibrary (Roche applied science) and LightCycler TaqMan Master (Roche applied science). Cycling profiles for real-time PCR were pre-incubated for 10 sec at 95° C., and carried out 55 cycles of 5 sec at 95° C., 20 sec at 60° C., and 2 sec at 72° C. Fluoresce was acquired on each elongation step during amplification and analyzed with the Light Cycler Software 4.05. Significantly, brain-derived neurotrophin factor gene (BDNF) was found to be down-regulated while the levels of other genes such as TrkB, NT-3, HRPT, and Actb-1 remain unchanged. This correlates with the observation that expression levels of BDNF decreased with age as demonstrated in the 15-month and 28-month-old wild-type mice compared with that in the younger mice (
A summary of the aging-related phenotypes in Cisd2−/− mice was provided in Table 1. These mutant mice exhibited a premature aging phenotype with 100% penetrance for both sexes using either a C57BL/6 (B6) or a 129Sv/B6 mixed background.
§In the wild-type control mice, the median lifespan is 109 wk; minimum lifespan is 72 wk; maximum lifespan is 132 wk.
#Data obtained from male mice
$Data collected from the adipose layer of cutaneous tissue.
The observation of premature aging phenotypes involving muscle degeneration prompted a detailed examination of the tissue ultrastructure of the homozygous knockout mice. A TEM study revealed that mitochondrial degeneration occurred in the axons of sciatic nerves, brain cells (
Importantly, these mitochondrial abnormalities, involving destruction of mitochondria, myelin sheath disintegration and axonal lesions, were already present to a certain extent in 2-week old Cisd2−/− mice (
In addition, it has been reported that starvation could induce muscle autophagy. To test this possibility, the present invention measured the metabolic indices including intake of food and water and generation of urine and stool. The results of the present invention revealed no significant difference in these metabolic indices between Cisd2−/− and wild-type mice at 6-week old (
The annotated characteristics of Cisd2 protein were very similar to Cisd1, which is an outer mitochondrial membrane protein (
Previously a report showed that the FLAG-tagged CISD2 protein colocalized with the ER marker calnexin in the transfected mouse P19 and human HEK293 cells. The present invention sought to determine if there was a small portion of the Cisd2 protein sorted into the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) using subcellular fractions prepared from skeletal muscles of 11 wild-type mice. The data of the present invention indeed revealed a weak signal indicating the presence of Cisd2 protein in the post-mitochondrial supernatant and this colocalized with the ER markers in the microsomal fractions. The ratio of the Cisd2 protein present in the mitochondria versus ER was estimated to be about 5.8:1 (
Mitochondria are the cellular energy factories that generate ATP via oxidative phosphorylation. To investigate whether the mitochondrial degeneration detected in this study has a direct functional consequence leading to a respiratory dysfunction, the present invention assessed mitochondrial aerobic respiration using isolated mitochondria prepared from skeletal muscle. This was done by measuring the oxygen consumption after stimulating the mitochondria with glutamate-malate and ADP to activate the respiratory chain reactions. The results of the present invention revealed a significant decrease in the oxygen consumption and the respiratory control ratio in the Cisd2−/− mitochondria (
In order to evaluate the usefulness of Cisd2−/− mice as an animal model for WFS2 and gain insight into the mechanistic basis of WFS2 pathogenesis, the present invention compared the clinical manifestations of this disease and the phenotype of Cisd2−/− mice. WFS2 is a clinically heterogeneous disease; only juvenile-onset diabetes mellitus and optic atrophy are necessary criteria for WFS2 diagnosis. Importantly, Cisd2−/− mice exhibited a progressive neurodegenerative phenotype that included optic nerve defects (
#Clinical features are based on Barrett, T. G. & Bundey, S. E. Wolfram (DIDMOAD) syndrome. 1997. J. Med. Genet. 34, 838-841.
Resveratrol (30 mg/kg/day) was administered by oral to Cisd2 knockout mice from 4- to 12-week old. The body weight of the Cisd2 knockout mice was analyzed after resveratrol treatment and comparing to 4-week old wild-type mice (
Results in
Insofar as the description above and the accompanying drawing disclose any additional subject matter that is not within the scope of the single claim below, the inventions are not dedicated to the public and the right to file one or more applications to claim additional inventions is reserved.
Although a very narrow claim is presented herein, it should be recognized the scope of this invention is much broader than presented by the claim. It is intended that broader claims will be submitted in an application that claims the benefit of priority from this application.
This application is a Continuation-in-part of the pending U.S. patent application Ser. No. 12/481,042 filed on Jun. 9, 2009, which is a Continuation-in-part U.S. patent application Ser. No. 11/866,374 of which claims priority U.S. Application No. 60/849,089 filed on Oct. 3, 2006, all of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60849089 | Oct 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12481042 | Jun 2009 | US |
| Child | 13041821 | US | |
| Parent | 11866374 | Oct 2007 | US |
| Child | 12481042 | US |
