Abstract:
A method of distinguishing a brown adipose cell from a white adipose cell. In one embodiment the method includes measuring the expression level of one or more genes in an adipose cell; comparing the measured expression levels to a control, and correlating the expression level of the one or more genes to an identity as a white adipose cell or a brown adipose cell. In one embodiment the one or more genes are selected from the genes listed in FIG.  4 C. In another aspect the invention relates to a method of differentiating an adipose stem cell. In one embodiment the method includes inducing differentiation of an adipose stem cell in vitro; and distinguishing the differentiated stem cell. In another embodiment the inducing is performed by contacting the adipose stem cell with a brown adipose cell differentiation media.

Description:
RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. Provisional applications 61/756,857 filed Jan. 25, 2013 and 61/757,900 filed Jan. 29, 2013; the contents of each are herein incorporated in their entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates generally to the field of cell culture and more specifically to the field of determining cell type. 
       BACKGROUND 
       [0003]    Brown adipose tissue (BAT) plays a key role in the evolutionarily conserved mechanisms underlying energy homeostasis in mammals. It is characterized by fat vacuoles 5-10 microns in diameter and expression of uncoupling protein 1 (UCP1), central to the regulation of thermogenesis. In the human newborn, depots of BAT are typically grouped around the vasculature and solid organs. These depots maintain body temperature during cold exposure by warming the blood before its distribution to the periphery. They also ensure an optimal temperature for biochemical reactions within solid organs. BAT had been thought to involute throughout childhood and adolescence. Recent studies, however, have confirmed the presence of active brown adipose tissue in adult humans with depots residing in cervical, supraclavicular, mediastinal, paravertebral and suprarenal regions. While human pluripotent stem cells have been differentiated into functional brown adipocytes in vitro and inducible brown adipocyte progenitor cells have been identified in murine skeletal muscle and white adipose tissue, metabolically active brown adipose tissue derived stem cells have not been identified in adult humans to date. 
         [0004]    The present invention addresses this issue. 
       SUMMARY OF THE INVENTION 
       [0005]    In one aspect the invention relates to a method of distinguishing a brown adipose cell from a white adipose cell. In one embodiment the method includes measuring the expression level of one or more genes in an adipose cell; comparing the measured expression levels to a control, and correlating the expression level of the one or more genes to an identity as a white adipose cell or a brown adipose cell. In one embodiment the one or more genes are selected from the genes listed in  FIG. 4C . In another embodiment an increase in expression of one or more of the following genes as compared to the control is indicative that the adipose cell is a brown adipose cell: ACACB, ADRB2, FGF10, KLF15, LIPE, NR1H3, CIDEC, ELOVL3, INHBB, PPARGC1A, and UCP1. In yet another embodiment an increase in expression of LEP as compared to the control is indicative that the adipose cell is a white adipose cell. In still yet another embodiment the method measures the expression level by quantifying transcript levels. 
         [0006]    In one embodiment the method measures the levels of at least two, at least three, at least four, at least five, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 genes. In another embodiment the method measures the levels of any one of ELOVL3, INHBB, PPARGC1A, or UCP1. In yet another embodiment the method measures the levels of any two of ELOVL3, INHBB, PPARGC1A, or UCP1. In still yet another embodiment the method measures the levels of any three of ELOVL3, INHBB, PPARGC1A, or UCP1. In one embodiment the method measures the levels of ELOVL3, INHBB, PPARGC1A, and UCP1. 
         [0007]    In another aspect the invention relates to a method of differentiating an adipose stem cell. In one embodiment the method includes inducing differentiation of an adipose stem cell in vitro; and distinguishing the differentiated stem cell. In another embodiment the inducing is performed by contacting the adipose stem cell with a brown adipose cell differentiation media. In yet another embodiment the inducing is performed by contacting the adipose stem cell with FNDC5. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0008]    The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in anyway. 
           [0009]      FIG. 1(A)  is a series of diagrams of flow cytometry results of undifferentiated brown adipose derived stem cells; 
           [0010]      FIG. 1(B)  is a photomicrograph of biopsied mediastinal brown adipose depots that demonstrate multiocular lipid morphology and UCP1 staining specific to brown adipose tissue; 
           [0011]      FIG. 1(C)  is a karyotype analysis of passage 10 brown adipose derived stem cells; 
           [0012]      FIG. 2  is a set of flow cytometry results of TMEM26 and CD137 of brown and white adipose derived stem cells; 
           [0013]      FIG. 3(A)  is a Western blot of cells 21 days post FNDC5 induction; 
           [0014]      FIG. 3(B)  is a photomicrograph of Alcian blue stained brown adipose derived stem cells directionally differentiated into chondrocytes; 
           [0015]      FIG. 3(C)  is a photomicrograph of fatty acid binding protein 4 (FABP4) immunocytochemistry of brown adipose derived stem cells induced to undergo white adipogenesis; 
           [0016]      FIG. 3(D)  is a photomicrograph of Alizarian red stained brown adipose derived stem cells induced to undergo osteogenesis; 
           [0017]      FIG. 4(A)  is a SEM of brown adipose derived stem cells cultured on porous extracellular matrix scaffolds; 
           [0018]      FIG. 4(B)  is a SEM of directionally differentiated brown adipocytes on scaffolds; 
           [0019]      FIG. 4(C)  is a transcriptional profile of brown adipose derived stem cells differentiated into brown and white adipocytes; 
           [0020]      FIG. 4(D)  is a measure of fatty acid uptake of brown fat differentiated brown adipose derived stem cells at 7, 14 and 21 days post differentiation; 
           [0021]      FIG. 4(E)  is a measure of functional mitochondrial respiration assay of brown adipose derived stem cells differentiated into brown adipocytes at 7, 14 and 21 days post differentiation; 
           [0022]      FIG. 5(A)  is a graph of expression levels of MSC associated genes; 
           [0023]      FIG. 5(B)  is a graph of expression levels of MSC specific genes; and 
           [0024]      FIG. 5(C)  is a graph of expression levels of sternness genes. 
       
    
    
     DETAILED DESCRIPTION 
       [0025]    Briefly, for this study, human adipose tissues were biopsied and analyzed with immunohistochemistry and primary cell isolation. Primary cells isolated from adipose explants were expanded and their growth kinetics, karyotyping, flow cytometry and immunocytochemistry were determined. Passage-2 cells were directionally differentiated into osteogenic, chondrogenic, white adipogenic and brown adipogenic lineages on plastic and also differentiated into brown adipocytes on porous extracellular matrix scaffolds. Differentiation was confirmed by Western blot, immunohistochemistry, cytochemistry, scanning electron microscopy (SEM), and quantitative real-time PCR. Functional brown fat differentiation was confirmed by fatty acid uptake and mitochondrial respiration, as measured by the oxygen consumption rate (OCR). 
       Methods 
     Mediastinal Adipose Tissue Procurement 
       [0026]    Mediastinal adipose tissues were obtained from 54 patients undergoing cardiac surgery. The group included 44 males and 10 females and had a mean ±SE age 72.4±12 yr. (range 28-84 yr.). 
       Derivation of Mediastinal Adipose Derived MSCs 
       [0027]    The excised tissue was cut into 3 mm pieces and explanted onto a 6 well dish and grown in DMEM low glucose, 10% XcytePL™ Supplement (JadiCell, Phoenix, Ariz.), 1X Glutamax, and 1X MEM-NEAA (Life Technologies, Carlsbad, Calif.) and cultured in 5% CO 2 /37° C. 
       RNA Analysis 
       [0028]    RNA was isolated and DNaseI treated using the RNAqueous-4PCR Kit (Life Technologies AM1914 (Life Technologies, Carlsbad, Calif.)) per manufacturer&#39;s protocol. 
         [0029]    First strand cDNA was synthesized using the RI&#39; First Strand Kit (SABiosciences 330401) (SABiosciences, Valencia, Calif.) per manufacturer&#39;s protocol. 
         [0030]    PCR was carried out on RT 2  Profiler PCR Arrays using RT 2 SYBR Green qPCR Mastermix (SABiosciences 330521) in an Eppendorf Mastercycler ep realplex 4 pcr machine (Eppendorf, Hauppauge, N.Y.) per manufacturer&#39;s protocol. 
         [0031]    The following RT 2  Profiler PCR Arrays and individual gene primers were used:
   Human Adipogenesis (SABiosciences PAHS-049A)   Human Mesenchymal Stem Cells (SABiosciences PAHS-082A)   RT 2  qPCR Primer Assay for CIDEC(SABiosciences PPH18299E)   RT 2  qPCR Primer Assay for COX8A (SABiosciences PPH20233A)   RT 2  qPCR Primer Assay for CYC1(SABiosciences PPH00724A)   RT 2  qPCR Primer Assay for CYFIP2 (SABiosciences PPH14474E)   RT 2  qPCR Primer Assay for DPT (SABiosciences PPH10191A)   RT 2  qPCR Primer Assay for ELOVL3(SABiosciences PPH16532A)   RT 2  qPCR Primer Assay for INHBB(SABiosciences PPH01917A)   RT 2  qPCR Primer Assay for LHX8(SABiosciences PPH19135A)   RT 2  qPCR Primer Assay for NDUFA11(SABiosciences PPH19207A)   RT 2  qPCR Primer Assay for NDUFA13(SABiosciences PPH60028A)   RT 2  qPCR Primer Assay for PMP22(SABiosciences PPH02152E)   RT 2  qPCR Primer Assay for GJA1(SABiosciences PPH02781E)   RT 2  qPCR Primer Assay for MYH7(SABiosciences PPH00044E)   RT 2  qPCR Primer Assay for NKX2-5(SABiosciences PPH02462A)   RT 2  qPCR Primer Assay for TNNT2(SABiosciences PPH02619A)   RT 2  qPCR Primer Assay for B2M(SABiosciences PPH01094E)   RT 2  qPCR Primer Assay for HPRT1(SABiosciences PPH01018B)   RT 2  qPCR Primer Assay for RPL13A(SABiosciences PPH01020B)   
 
         [0052]    Delta delta (ΔA) Ct based fold-change calculations were performed using the RT 2  Profiler PCR Array Data Analysis Web Portal version 3.5 provided by SABiosciences at: http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php 
         [0053]    Total RNA was purified and DNase-treated from individual wells of 24-well plates using the RNAqueous-Micro kit (Ambion AM1931(Life Technologies, Carlsbad, Calif.)) per manufacturer&#39;s protocol. 100 ng of each sample was reverse transcribed and pre-amplified using the RT 2  PreAMP cDNA Synthesis Kit (SABiosciences 330241) (SABiosciences, Valencia, Calif.). The preamplified product was then amplified using the RT 2  SYBR Green/ROX qPCR Master Mix (SABiosciences PA-012) (SABiosciences, Valencia, Calif.). Experiments were done in triplicate and data was analysed by the delta delta (ΔA) Ct method. The control gene used was HPRT1. 
       Immunocytochemistry 
       [0054]    Cells were fixed with 4% paraformaldehyde. After blocking, cells were incubated with primary antibody diluted in 5% donkey serum. After washing cells were incubated with secondary antibody and counterstained with DAPI (Molecular Probes (Life Technologies, Carlsbad, Calif.)). For negative controls, incubation without primary antibody and with corresponding specific non-immune immunoglobulin (EMD Millipore, Billerica, Mass.) was used. 
       Flow Cytometry 
       [0055]    Directly conjugated antibodies used: HLA-DP DQ DR (BD Biosciences,), CD90, LIN, CD166, STRO-1, SSEA-4, CD44, CD106, CD73, CD117, CD105, HLA-ABC, CD86, CD63, CD9, CD80 (Biolegend, San Diego, Calif.), CD45, CD133, and CD34 (Miltenyi Biotech, Bergisch Gladbach, Germany). After staining, the cells were fixed and analyzed using a FACSCanto II analyzer (BD Biosciences, Franklin Lakes, N.J.). 
       Adipogenic Differentiation 
       [0056]    Cells were plated in 6-well dishes at a density of 50,000 cells/well. White or brown adipogenesis differentiation medium was added. For brown adipogenesis, FNDC5 was added 6 days post induction. Fatty acid binding protein 4 (FABP4) immunocytochemistry and 0.3% Oil Red O (Sigma Aldrich, St. Louis, Mo.) was used for staining to detect intracellular lipid accumulation (Data not shown). 
       Osteogenic Differentiation 
       [0057]    Cells were plated in 6 well dishes at a density of 50,000 cells/well. StemPro® Osteogenesis Differentiation medium ((Life Technologies, Carlsbad, Calif.)) was added. 2% Alizarian Red S (Sigma Aldrich, St. Louis, Mo.) was used for staining to detect de novo formation of bone matrix. 
       Chondrogenic Differentiation 
       [0058]    500,000 cells/15 ml tube were pelleted and induced with StemPro® Chondrogenesis Differentiation medium ((Life Technologies, Carlsbad, Calif.)). 1% Alcian Blue (Sigma Aldrich, St. Louis, Mo.) was used to detect sulfated glycosaminoglycans. 
       Fatty Acid Uptake Analysis 
       [0059]    Analysis began with the replacement of growth media with HBSS Buffer with 20 mM HEPES and 0.2% fatty free BSA. Cells were placed in the incubator for 1.5 h, QBT Fatty Acid Uptake (Molecular Devices, Sunnyvale, Calif.) media was added to the wells and fluorescence was analyzed every minute in a Bio-Tek Synergy HT (Bio Tek, Winooski, Vt.). 
       Cellular Respiration and Glycolysis Analysis 
       [0060]    The oxygen consumption rate (OCR) was performed using a Seahorse Bioscience XF-24 instrument (Seahorse Bioscience, Billerica, Mass.). Analysis was performed by replacing the growth media with XF assay media and incubating in a CO 2  free chamber for 1 h. The XF Cell Mito Stress Test simultaneously analyzed basal respiration, ATP turnover, proton leak, spare respiratory capacity and glycolysis. 
       Transmission Electron Microscopy 
       [0061]    Samples were fixed and embedded for routine TEM. They were then examined on an FEI Tecnai T-12 (FEI Hillsboro, Oreg.) at 120 KV. 
       Scanning Electron Microscopy 
       [0062]    Scaffolds were fixed and post fixed in 2% osmium tetroxide, dehydrated through a series of ethanol washes, dried with hexamethyldisilazane. Scaffolds were then sputter coated with gold and imaged with a scanning electron microscope under high vacuum. 
       Results 
       [0063]      FIG. 1(A)  shows flow cytometry of undifferentiated brown adipose derived stem cells. The cells expressed CD44, CD105, CD166, and CD90 and were negative for hematopoietic markers CD34, CD45, and HLA-DR.  FIG. 1(B)  is a photomicrograph of biopsied mediastinal brown adipose depots demonstrate multiocular lipid morphology and UCP1 staining specific to brown adipose tissue.  FIG. 1(C)  is a karyotype analysis of passage 10 brown adipose derived stem cells. 
         [0064]      FIG. 2  depicts the flow cytometry results of TMEM26 and CD 137 of brown and white adipose derived stem cells. Brown adipose derived stem cells express higher levels of TMEM26 and CD137. 
         [0065]      FIG. 3(A)  is a Western blot 21 days post FNDC5 induction. Lane 1 holds brown adipose derived stem cells directionally differentiated into brown adipocytes. Lane 2 holds undifferentiated brown adipose derived stem cells.  FIG. 3(B)  is a photomicrograph of Alcian blue stained brown adipose derived stem cells directionally differentiated into chondrocytes.  FIG. 3(C)  is a photomicrograph of the fatty acid binding protein 4 (FABP4) immunocytochemistry of brown adipose derived stem cells induced to undergo white adipogenesis.  FIG. 3(D)  is a photomicrograph of Alizarin red stained brown adipose derived stem cells induced to undergo osteogenesis. 
         [0066]      FIG. 4(A)  is a SEM of brown adipose derived stem cells cultured on porous extracellular matrix scaffolds.  FIG. 4(B)  SEM of directionally differentiated brown adipocytes on scaffolds.  FIG. 4(C)  is a transcriptional profile of brown adipose derived stem cells differentiated into brown and white adipocytes.  FIG. 4(D)  is a graph of fatty acid uptake of brown fat differentiated brown adipose derived stem cells at 7, 14 and 21 days post differentiation.  FIG. 4(E)  is a graph of the results of a functional mitochondrial respiration assay of brown adipose derived stem cells differentiated into brown adipocytes at 7, 14 and 21 days post differentiation. 
         [0067]    In  FIG. 4(C) , profiled genes are listed according to their standard abbreviation (NCBI gene profile):
       ACACB: acetyl-CoA carboxylase beta   ADIG: adipogenin   ADIPOQ: Adiponectin   ADRB2: adrenoceptor beta 2, surface   AGT: angiotensinogen   BMP4: bone morphogenetic protein 4   CCND1: cyclin D1   CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha   CFD: complement factor D (adipsin)   DKK1: Dickkopf1   DLK1: delta-like 1 homolog   E2F1: E2F transcription factor 1   FABP4: fatty acid binding protein 4   FASN: Fatty acid synthase   FGF1: fibroblast growth factor 1   FGF10: fibroblast growth factor 10   FGF2: fibroblast growth factor 2   FOXO1: forkhead box O1   GATA2: GATA binding protein 2   HES1: hairy and enhancer of split 1   IRS2: insulin receptor substrate 2   KLF15: Kruppel-like factor 15   KLF2: Kruppel-like factor 2   LEP: Leptin   LIPE: hormone-sensitive lipase   LMNA: lamin A/C   LPL: lipoprotein lipase   NR1H3: nuclear receptor subfamily 1, group H, member 3   PPARG: peroxisome proliferative activated receptor, gamma   SLC2A4: solute carrier family 2 (facilitated glucose transporter)   SREBF1: sterol regulatory element binding transcription factor 1   TSC22D3: TSC22 domain family, member 3   VDR: Vitamin D3 receptor   WNT10B: wingless-type MMTV integration site family, member 10B   WNT5B: wingless-type MMTV integration site family, member 5b   CIDEC: cell death-inducing DFFA-like effector c   CYFIP2: Cytoplasmic FMR1-interacting protein 2   DIO2: deiodinase, iodothyronine, type II   DPT: Dermatopontin   ELOVL3: Elongation of very long chain fatty acids protein 3   FOXC2: forkhead box C2   INHBB: inhibin, beta B   INSR: insulin receptor   PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1   UCP1: uncoupling protein 1.       
 
         [0113]    Table 1 is a list of genes expressed by brown and white MSC as measured against a standard along with a measure of their expression relative to the standard. Thus for example The expression of the gene ANXA5 is 1.178 fold higher in brown than in the standard. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Gene 
                 Brown 
                 White 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 ANXA5 
                 1.178 
                 0.150 
               
               
                   
                 BDNF 
                 2.573 
                 0.555 
               
               
                   
                 BGLAP 
                 1.214 
                 0.180 
               
               
                   
                 BMP7 
                 −1.192 
                 0.421 
               
               
                   
                 COL1A1 
                 −1.206 
                 0.203 
               
               
                   
                 CSF2 
                 −1.019 
                 0.315 
               
               
                   
                 CSF3 
                 −1.143 
                 0.523 
               
               
                   
                 CTNNB1 
                 1.243 
                 0.190 
               
               
                   
                 EGF 
                 1.240 
                 0.510 
               
               
                   
                 FUT1 
                 5.110 
                 2.330 
               
               
                   
                 GTF3A 
                 1.275 
                 0.195 
               
               
                   
                 HGF 
                 1.613 
                 0.295 
               
               
                   
                 ICAM1 
                 −3.211 
                 0.360 
               
               
                   
                 IFNG 
                 −1.059 
                 0.239 
               
               
                   
                 IGF1 
                 −2.822 
                 3.061 
               
               
                   
                 IL10 
                 −1.982 
                 4.268 
               
               
                   
                 IL1B 
                 4.532 
                 0.720 
               
               
                   
                 IL6 
                 13.056 
                 2.485 
               
               
                   
                 ITGB1 
                 1.200 
                 0.190 
               
               
                   
                 KITLG 
                 −1.248 
                 0.290 
               
               
                   
                 MITF 
                 1.050 
                 0.220 
               
               
                   
                 MMP2 
                 −1.709 
                 0.252 
               
               
                   
                 NES 
                 2.346 
                 0.455 
               
               
                   
                 NUDT6 
                 1.643 
                 0.120 
               
               
                   
                 PIGS 
                 1.067 
                 0.095 
               
               
                   
                 PTPRC 
                 1.347 
                 0.805 
               
               
                   
                 SLC17A5 
                 1.257 
                 0.215 
               
               
                   
                 TGFB3 
                 −1.125 
                 0.141 
               
               
                   
                 TNF 
                 1.729 
                 0.970 
               
               
                   
                 VEGFA 
                 1.502 
                 0.320 
               
               
                   
                 VIM 
                 −1.347 
                 0.292 
               
               
                   
                 VWF 
                 −1.382 
                 0.891 
               
               
                   
                 ALCAM 
                 4.327 
                 0.665 
               
               
                   
                 ANPEP 
                 −1.228 
                 0.125 
               
               
                   
                 BMP2 
                 1.035 
                 0.125 
               
               
                   
                 CASP3 
                 1.879 
                 0.150 
               
               
                   
                 CD44 
                 1.866 
                 0.305 
               
               
                   
                 ENG 
                 1.454 
                 0.145 
               
               
                   
                 ERBB2 
                 1.228 
                 0.250 
               
               
                   
                 FUT4 
                 1.272 
                 0.205 
               
               
                   
                 FZD9 
                 1.329 
                 0.650 
               
               
                   
                 ITGA6 
                 12.524 
                 2.395 
               
               
                   
                 ITGAV 
                 1.206 
                 0.165 
               
               
                   
                 KDR 
                 14.254 
                 5.895 
               
               
                   
                 MCAM 
                 1.431 
                 0.260 
               
               
                   
                 NGFR 
                 −1.102 
                 0.440 
               
               
                   
                 NT5E 
                 1.725 
                 0.225 
               
               
                   
                 PDGFRB 
                 −2.139 
                 0.100 
               
               
                   
                 PROM1 
                 0.010 
                 0.000 
               
               
                   
                 THY1 
                 −1.082 
                 0.115 
               
               
                   
                 VCAM1 
                 15.780 
                 9.470 
               
               
                   
                 FGF2 
                 1.643 
                 0.275 
               
               
                   
                 INS 
                 0.000 
                 0.000 
               
               
                   
                 LIF 
                 3.855 
                 0.805 
               
               
                   
                 POU5F1 
                 3.767 
                 0.925 
               
               
                   
                 SOX2 
                 0.000 
                 0.000 
               
               
                   
                 TERT 
                 0.000 
                 0.000 
               
               
                   
                 WNT3A 
                 0.000 
                 0.000 
               
               
                   
                 ZFP42 
                 2.240 
                 0.670 
               
               
                   
                   
               
             
          
         
       
     
         [0114]      FIG. 5  is a graph of the expression of MSC associated genes. MSC associated genes are genes that are generally found in all mesenchymal stem cells to some degree.  FIG. 6  is a graph of the expression of MSC specific genes. These genes are generally unique to mesenchymal stem cells.  FIG. 7  is a graph of the expression of Sternness genes. These genes generally are found in cells with more differentiation potential such as embryonic stem cells 
         [0115]    These results uniquely demonstrate a resident stem cell population within depots of brown adipose tissue from adult human mediastinum. Cells from this tissue exhibit multi-lineage potential with capacities to undergo osteogenesis, chondrogenesis and both brown and white adipogenesis. Directionally differentiated brown adipocytes exhibit a distinct morphology and gene expression profile, with functional properties characteristic of brown adipose tissue in vivo. These brown adipose-derived stem cells may offer a new target to activate and restore energy homeostasis in vivo for the treatment of obesity and related metabolic disorders. 
         [0116]    It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.