CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional Patent Application No. 63/256,847, filed Oct. 18, 2021, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Numbers HD106285 awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND

Uterine fibroids (UFs) are benign smooth muscle tumors that are a major cause of gynecologic dysfunction, such as menometrorrhagia and anemia, pelvic pressure and bulk symptoms, infertility, recurrent miscarriage, and preterm labor. UFs are the most common pelvic tumor, occurring in 80% of women. Uterine fibroids exhibit an extraordinary range of clinical presentations with lesions that routinely range from 5 mm to over 25 cm in size.

There is an ongoing need in the art to treat UFs.

BRIEF SUMMARY

In aspects, the present disclosure provides a method of treating or preventing a uterine fibroid in a female mammal, the method comprising, consisting essentially of, or consisting of administering to the female mammal an effective amount of an agent that modulates an N6-methyladenosine (m6A) regulator.

Additional aspects of the present disclosure are as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram that illustrates the N6-methyladenosine (m6A) pathway in the epitranscriptome.

FIGS. 2A-2B are a Western blot image of methyltransferase-like protein 3 (METTL3) and β actin levels in UFs and myometrium from fibroid containing uterus (MyoF) of humans (FIG. 2A) and an immunoblot analysis dot graph of the fold change in UF over MyoF of METTL3 levels (FIG. 2B). P indicates the patient (n=7), M indicates the MyoF tissue (n=7), F indicates the UFs (n=22).

FIGS. 3A-3F are a Western blot image of METTL3 and β actin levels in immortalized human uterine leiomyoma (HuLM) cells and immortalized uterine smooth muscle (UTSM) cells (FIG. 3A), a Western blot image of METTL3 and β actin levels in HuLM cells treated with scrambled or METTL3 shRNA (FIG. 3B), an immunoblot analysis dot graph of the fold change of METTL3 over actin in the scramble and METTL3 shRNA treated HuLM cells (FIG. 3C), a Western blot image of Bax, Bcl2, and β actin levels in HuLM cells treated with scrambled or METTL3 shRNA (FIG. 3D), an immunoblot analysis dot graph of the fold change in BAX and Bcl2 over β actin in HuLM cells treated with METTL3 shRNA (FIG. 3E), and a cell proliferation analysis bar graph showing the relative proliferation of HuLM cells treated with scrambled control shRNA (CTL) and METTL3 shRNA (KD) when cultured in the presence of a vehicle control (VEH) or Staurosporine (STS) (FIG. 3F).

FIGS. 4A-4B are a Western blot image of YT521-B homology domain containing protein 2 (YTHDC2) and β actin levels in UFs and MyoF tissue of humans (FIG. 4A) and an immunoblot analysis dot graph of the fold change in UF over MyoF of YTHDC2 levels (FIG. 4B). P indicates the patient (n=7), M indicates the MyoF tissue (n=7), F indicates the UFs (n=22).

FIG. 5 is a graph of the risk of leiomyoma in Eker rats in the absence or presence of an estrogen treatment.

FIGS. 6A-6B are a Western blot image of METTL3, YTHDC2, and YT521-B homology domain N6-methyladenosine RNA binding protein 2 (YTHDF2) and β actin levels in UFs (F) and MyoF (M) tissue of late adult stage Eker rats (n=5) (FIG. 6A) and an immunoblot analysis dot graph of the fold change in UF over MyoF of METTL3, YTHDC2, and YTHDF2 levels (FIG. 6B). Late adult stage Eker rats are 11-15 months old.

FIG. 7 is a quantitative PCR analysis dot graph showing fold change of UF (n=5) over MyoF (n=5) expression level of the m6A regulators METTL3, methyltransferase-like protein 14 (METTL14), human AlkB homolog 5 (ALKBHS), fat mass and obesity-associated protein (FTO), YT521-B homology domain containing protein 1 (YTHDC1), YTHDC2, YT521-B homology domain N6-methyladenosine RNA binding protein 1 (YTHDF1), and YTHDF2 in Eker rats.

FIGS. 8A-8B are illustrations of the chemical structures of the m6A inhibitors DAC51 (FIG. 8A) and STM2457 (FIG. 8B).

FIG. 9 is a cell proliferation bar graph showing the relative cell numbers in the absence or presence of the METTL3 inhibitor, STM2457, at concentrations between 0-10 μM, in HuLM cells.

DETAILED DESCRIPTION

In aspects, the present disclosure provides a method of treating or preventing a uterine fibroid in a female mammal, the method comprising, consisting essentially of, or consisting of administering to the female mammal an effective amount of an agent that modulates an N6-methyladenosine (m6A) regulator.

As used herein, a “uterine fibroid” (UF), is a benign tumor of the uterus that consists of a mass or population of smooth muscle cells and connective tissue that grows, usually slowly, within the uterine wall. Epidemiologic studies demonstrate that UFs, also known as leiomyomas, initially form after menarche. It is suspected that fibroid growth is due to a monoclonal, deregulated proliferation of uterine smooth muscle myometrial cells. The primary tumor cell type resulting from the growth of the fibroid are derived from myometrial cells.

UFs have a high accumulative incidence. UFs are one of the most common tumors. Complications arising from uterine fibroids account for approximately a third of all hysterectomies performed in the U.S., and are associated with high morbidity from uterine bleeding and pain. By age 50 approximately 75% of women have developed UFs. A significant number of those with UFs suffer from debilitating pelvic pain, heavy and prolonged bleeding, which may lead to anemia and iron deficiency, bowel and bladder dysfunction, and infertility. UFs also cause symptoms such as low back pain, urinary frequency and urgency, pain during intercourse (dyspareunia), can cause pre-term labor, and have a negative impact on fertility (due to cavity distension, and alteration of endometrial receptivity and sexual function).

The terms “treat,” “treating,” “treatment,” “therapeutically effective,” etc. used herein do not necessarily imply 100% or complete treatment, etc. Rather, there are varying degrees, which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the agent that modulates an m6A regulator and methods can provide any amount of any level of treatment. Furthermore, the treatment provided by the disclosed method can include the treatment of one or more conditions or symptoms of the disease or condition being treated.

The disclosed methods comprise using an effective amount of an agent that modulates an m6A regulator. An “effective amount” means an amount sufficient to show a meaningful benefit. A meaningful benefit includes, for example, detectably treating, relieving, or lessening one or more symptoms of UFs; inhibiting, arresting development, preventing, or halting further development of UFs; reducing the size and/or mass of UFs; reducing the severity of UFs; preventing UFs from occurring in a subject at risk thereof but yet to be diagnosed. The meaningful benefit observed can be to any suitable degree (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). In aspects, one or more symptoms are prevented, reduced, halted, or eliminated subsequent to administration of an agent that modulates an m6A regulator as described herein, thereby effectively treating the disease to at least some degree.

One skilled in the art will recognize that dosage will depend upon a variety of factors, including the age, condition or disease state, predisposition to disease, genetic defect or defects, and body weight of the subject. The size of the dose will also be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular active agent and the desired effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.

The mammal may be any suitable mammal. Mammals include, but are not limited to, the order Rodentia, such as mice, and the order Lagomorpha, such as rabbits. The mammal can be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammal can be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perissodactyla, including Equines (horses). The mammal can be of the order Primates, Cebids, or Simioids (monkeys) or of the order Anthropoids (humans and apes). In aspects, the mammal is human.

The epitranscriptome includes all the biochemical modifications of the RNA within a cell. Among these post-transcriptional RNA modifications, m6A is the abundant, dynamic, and reversible modification involved in many biological events and diseases.

As used herein, the term “m6A regulator” is any molecule that regulates m6A modifications to RNA. Without wishing to be bound by theory, m6A regulators can be further categorized as “erasers,” “readers,” and “writers.” As used herein, an “eraser” is defined as a category of enzymes that demethylate m6A. m6A erasers include fat mass and obesity-associated protein (FTO) and human AlkB homolog 5 (ALKBHS). As used herein, a “reader” is defined as a protein that recognizes and binds to m6A. m6A readers include YT521-B homology domain containing proteins (YTHDC) such as YTHDC1 and YTHDC2, YT521-B homology domain N6-methyladenosine RNA binding proteins (YTHDF) such as YTHDF1, YTHDF2, and YTHDF3, eukaryotic initiation factor 3 (eIF3), fragile X messenger riboprotein 1 (FMR1), heterogeneous nuclear ribonucleoproteins (HNRNPs) such as HNRNPC, HNRNPG, and HNRNPA2B1, and insulin-like growth factor 2 mRNA-binding proteins (IGF2BP) such as IGF2BP1, IGF2BP2, and IGF2BP3. As used herein, a “writer” is defined as an m6A methyltransferase complex that post-transcriptionally adds the m6A mark. The m6A methyltransferase complex include core subunits such as methyltransferase-like 3 (METTL3), which catalyzes the methyl transfer, and methyltransferase-like 14 (METTL14), which also methylates adenosine residues at the N(6) position of some mRNAs. The m6A methyltransferase complex also includes adaptor subunits such as RNA binding motif protein 15 (RBM15), Wilms' tumor 1-associating protein (WTAP), vir-like m6A methyltransferase associated (VIRMA), E3 ubiquitin-protein ligase Hakai (HAKAI), also known as casitas B-lineage lymphoma-transforming sequence-like protein 1 (CBLL1), and zinc finger CCCH-type containing protein 13 (ZC3H13). These adaptors play an important role in targeting the “writers” to distinct sets of genes in the chromatin, resulting in transcript-specific m6A methylation.

In aspects the agent that modulates an m6A regulator is an antibody. In aspect the agent is a lentivirus. Examples of lentiviruses that modulate an m6A regulator include, but are not limited to: the shMETTL3 lentivirus from Origene that inhibits through knockdown METTL3. The amount (e.g., therapeutically effective amount) of a lentivirus that modulates an m6A regulator suitable for administration depends on, e.g., the particular route of administration and the weight of the mammal to be treated. Several doses can be provided over a period of days. In aspects the agent that modulates an m6A regulator is the shMETTL3 lentivirus.

In aspects the agent that modulates an m6A regulator is a m6A inhibitor such as, but not limited to: inhibitory RNAs (RNA interference), the FTO inhibitor DAC51, and the METTL3 inhibitor STM2457. The amount (e.g., therapeutically effective amount) of an m6A inhibitor that modulates an m6A regulator suitable for administration depends on, e.g., the particular route of administration and the weight of the mammal to be treated. Several doses can be provided over a period of days. In aspects the agent that modulates an m6A regulator is the METTL3 inhibitor STM2457.

In aspects the m6A regulator is an m6A writer. Examples of m6A writers include, but are not limited to: METTL3, METTL14, RBM15, WTAP, VIRMA, HAKAI, also known as, CBLL1, and ZC3H13.

In aspects the m6a regulator is the m6A writer METTL3. In aspects the agent that modulates the m6A regulator is a lentivirus and the m6A regulator is METTL3. The amount (e.g., therapeutically effective amount) of a lentivirus that modulates METTL3 suitable for administration depends on, e.g., the particular route of administration and the weight of the mammal to be treated. In aspects, the amount of lentivirus that modulates an m6A regulator can be 50 multiplicity of infection (MOI). Several doses can be provided over a period of days.

In aspects the m6a regulator is an m6A eraser. Examples of m6A erasers include, but are not limited to: FTO and ALKBHS. In aspects the m6A eraser is FTO.

The following includes certain aspects of the disclosure.

1. A method of treating or preventing a uterine fibroid in a female mammal, the method comprising administering to the female mammal an effective amount of an agent that modulates an N6-methyladenosine (m6A) regulator.

2. The method of aspect 1, wherein the agent is an antibody.

3. The method of aspect 1, wherein the agent is a lentivirus.

4. The method of aspect 1, wherein the agent is STM2457.

5. The method of aspect 1, wherein the m6A regulator is an m6A writer.

6. The method of aspect 5, wherein the m6A writer is methyltransferase-like 3 (METTL3).

7. The method of aspect 2, wherein the m6A regulator is an m6A writer.

8. The method of aspect 7, wherein the m6A writer is methyltransferase-like 3 (METTL3).

9. The method of aspect 3, wherein the m6A regulator is an m6A writer.

10. The method of aspect 9, wherein the m6A writer is methyltransferase-like 3 (METTL3).

11. The method of aspect 10, wherein the lentivirus knocks down the METTL3.

12. The method of aspect 1, wherein the lentivirus is the shMETTL3 lentivirus.

13. The method of aspect 1, wherein the m6A regulator is an m6A eraser.

14. The method of aspect 13, wherein the m6A eraser is fat mass and obesity-associated protein (FTO).

15. The method of aspect 2, wherein the m6A regulator is an m6A eraser.

16. The method of aspect 15, wherein the m6A eraser is fat mass and obesity-associated protein (FTO).

17. The method of aspect 3, wherein the m6A regulator is an m6A eraser.

18. The method of aspect 17, wherein the m6A eraser is fat mass and obesity-associated protein (FTO).

It shall be noted that the preceding are merely examples of aspects of the disclosure. Other exemplary aspects are apparent from the entirety of the description herein. It will also be understood by one of ordinary skill in the art that each of these aspects may be used in various combinations with the other aspects provided herein.

The following examples further illustrate aspects of the disclosure, but, of course, should not be construed as in any way limiting its scope.

Example 1

This example demonstrates aberrant expression of m6A regulators and their role in human UFs.

Materials and Methods

UF Tissue Sample Collection

The UF tissues were obtained from the University of Chicago Tissue Bank.

Approval from the Institutional Review Board (#20-1414) at the University of Chicago was obtained for the retrospective chart review of UF patients. Informed consent was obtained from all the patients participating in the study before surgery. The cases with an initial diagnosis of UF at University of Chicago Hospital were reviewed, and the diagnosis was confirmed by H&E evaluation and immunohistochemistry. A total of nine cases with UFs were used and human UFs (n=22) and matched myometrium tissue (n=7) were collected at the time of hysterectomy.

Western Immunoblot

Cells were collected and lysed in RIPA lysis buffer with protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, Mass., USA), and the protein was quantified using the Bradford method (Bio-Rad Protein Assay kit). The antibodies used were METTL3 (ab195352, Abcam), YTHDC1 (ab122340, Abcam), YTHDC2 (35440, Cell Signaling), YTHDF2 (ab220163, Abcam), BCL-2 (ab182858, Abcam), FN (26836, Cell Signaling), PCNA (GTX100539, Genetex). The antigen-antibody complex was detected with Trident Femto Western HRP substrate (GeneTex, Irvine, Calif., USA). Specific protein bands were visualized using ChemiDoc XRS p molecular imager (Bio-Rad, Hercules, Calif., USA).

Cell Culture

The immortalized human leiomyoma cell line (HuLM) and immortalized human uterine smooth muscle cell line (UTSM) were cultured and maintained in phenol red-free, Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12. The cells were grown at 37° C. and 5% CO₂in an incubator with saturating humidity.

shRNA Knockdown of METTL3

METTL3 knockdown was performed in HuLM cells using shRNA delivered by lentivirus (Origene) to determine its role in uterine fibroids. The control group used scrambled shRNA (Origene). The HuLM cells were seeded in 12-well-dish (2.5×10⁵cells per well), and then incubate 18-20 hours at 37° C. in a humidified 5% CO₂incubator. The medium was removed, and a new medium with 50 MOI lentiviral particles and polybrene (8 ug/ml) was added. After 18-20 hours of culture, the medium was replaced with fresh pre-warmed complete culture medium. After 2 days, the cells were subjected to puromycin selection to obtain stable METTL13 knockdown cells.

Cell Proliferation Assay

Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 4×10⁴HuLM cells treated with scrambled shRNA or METTL3 shRNA per well were seeded into 12-well tissue culture plates with either vehicle (DMSO) or staurosporine (0.1 μM for 24 hours). This assay was performed three times in triplicate.

Quantitative Real Time PCR Analysis

Total RNA was isolated using Trizol reagent (Invitrogen, Calif. USA). The concentration of total RNA was determined using NanoDrop (Thermo Scientific, Waltham, Mass.). One microgram of total RNA from each sample was reverse transcribed to complementary DNA (cDNA) using the High-Capacity cDNA Transcription Kit (Thermo Scientific, Waltham, Mass.). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the messenger RNA (mRNA) expression of eight m6A regulators: writers (METTL3, METTL14), readers (YTHDC1,2; YTHDF1,2), and erasers (FTO, ALKBH5) in UF and matching MyoF tissue from Eker rats. The real-time PCR reactions were performed using CFX96 PCR instrument using SYBR Green Supermix (Bio-Rad, Hercules, Calif., USA). 18S was used as an internal control. The results are presented as relative gene expression using CFX MaestroTM. The assay was performed three times in triplicate.

Statistical Analysis

All experiments were conducted with at least three biological replicates. Comparisons between groups were made using student t-tests.

Results

Expression of m6A Regulators and their role in human UFs

Human UFs (n=22) and matched myometrium tissue (n=7) were collected at the time of hysterectomy from seven patients. Immunoblot analysis was performed on human uterine fibroids (n=22) and matching myometrium tissue (n=7) to determine METTL3 protein levels, using β Actin as a control. The student's t-test was used to determine the significant differences. The N6-methyladenosine writer METTL3 is significantly upregulated in UFs as compared to patient-matched myometrium as shown in FIGS. 2A-B. The METTL3 expression is also upregulated in cells from a uterine fibroid cell line (HuLM) as compared to those from a myometrial cell line (UTSM) as shown in FIG. 3A. METTL3 Knockdown using shRNA significantly downregulates METTL3 expression compared to scrambled shRNA controls, as shown in FIGS. 3B-C. Knockdown of METTL3 decreased the expression of BCL2, which plays a role in promoting cell survival and inhibiting the actions of pro-apoptotic proteins, as shown in FIGS. 3D-E. In addition, the knockdown of METTL3 increased the expression of BCL-2-associated X protein (BAX), a pro-apoptotic member of the BCL-2 family of genes, as shown in FIGS. 3D-E. Moreover, METTL3-knockdown cells, compared to control-knockdown cells, were characterized by significantly reduced proliferation and enhanced sensitivity to the apoptosis-inducing agent staurosporine, as shown in FIG. 3F.

Additional immunoblot analysis was performed in the same manner to determine the levels of YTHDC2 protein, a key reader in human UFs and myometrium tissues, as shown in FIG. 4A. The protein levels of YTHDC2 were significantly unregulated in human UFs compared to patient-matched MyoF, as shown in FIG. 4B.

These results demonstrate that m6A writer METTL3 protein is aberrantly expressed in human UFs. In addition to METTL3, other m6A regulators, such as YTHDC2, are also dysregulated in human UFs compared to matched MyoF tissue. Knockdown of METTL3 induces apoptosis in UF cells. UFs that have knocked down METTL3 levels are sensitive to apoptosis inducers, such as staurosporine, compared to surrounding myometrium.

Example 2

This example demonstrates UF development in Eker rats correlates with human UFs.

Materials and Methods

Eker Rat Husbandry

Female Eker rats [Long Evans; Tsc-2(Ek/+)] were obtained from an in-house Eker rat colony. All experiments using these animals were conducted in accordance with guidelines and provisions issued by the National Institutes of Health. Female Eker rats were raised to late adult stage, age 11-15 months. These rats were treated with hormones to induce UFs.

TABLE 1

Rat
Human

Presentation
High frequency (~65%)
High frequency

often multiple,
often multiple,

Histologically, primarily
Histologically, typical well-

epithelioid, also typical
differentiated smooth muscle,

and mixed
also epithelioid

Benign
Benign

Leiomyosarcoma very
Leiomyosarcoma very rare

infrequent

Hormone
Hormone-dependent
Hormone-dependent

Responsiveness
Expression of ER, PR
Expression of ER, PR

Response to SERMs
Response to GnRH agonists

Molecular
Pregnancy protective
Pregnancy protective

Alterations
TSC2
MED12

No specific chromosome
Characteristic translocation

aberrations
Aberrant expression of

Aberrant expression of
HMGA1 and A2

HMGA2

Results

UF Development in the Eker Rat Correlates with Human Disease

Eker rats harbor a germline insertion in the tuberous sclerosis 2 (Tsc2) gene that leads to its inactivation. Sixty percent of Eker rats carrying the TSC2 gene defect develop hormone-dependent UFs as shown in FIG. 5. Eker rats are therefore used as a model for human UF and related diseases. The availability of estrogen-responsive uterine fibroid-derived cell lines combined with the fact that spontaneous tumors develop with high frequency in female Eker rats has made this an animal model extremely useful for studies on uterine fibroids as shown in Table 1. Moreover, this model has the advantage to characterize the gene and environment interaction. Eker rats carrying a germline mutation in the tuberous sclerosis 2 (Tsc2) tumor suppressor gene spontaneously develop uterine fibroids with a frequency of 65% between ages 12 and 16 months. However, an early-life exposure to endocrine-disrupting chemicals such as diethylstilbestrol during the development of the uterus increases tumor-suppressor-gene penetrance to >90% and tumor multiplicity and size in genetically predisposed animals.

Eker rat UFs and adjacent MyoF tissues were collected at the late adult stage. The expression levels of METTL3, YTHDC2, and YTHDF2 are upregulated in UFs compared to matched MyoF in Eker rats, as shown in FIGS. 6A-B. Quantitative PCR was performed to determine the RNA expression of eight m6A regulators. As shown in FIG. 7, the expression levels of M6A regulators including METTL3, METTL14, ALKBHS, FTO, YTHDC2, YTHDF1, and YTHDF2 are upregulated in UFs as compared to matched MyoF tissues in Eker rats.

These results demonstrate that m6A writer METTL3 protein is aberrantly expressed in UFs from human and Eker rats. In addition to METTL3, other m6A regulators are also dysregulated in UFs from both human and Eker rats compared to matched MyoF tissues.

Example 3

This example demonstrates inhibiting m6A regulators decrease the proliferation of UF cells.

Materials and Methods

Cell Culture

The HuLM cell line was cultured and maintained in phenol red-free, Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12. The cells were grown at 37° C. and 5% CO₂in an incubator with saturating humidity.

Cell Proliferation Assay of METTL3 Inhibition

Cell proliferation was measured using trypan blue exclusion assay. The cells were cultured in the absence or presence of the METTL3 inhibitor STM2457 at a dose range from 1-10 μM for 24 hours. DMSO was used as a vehicle control. Cells were trypsinized, collected by centrifuge, and resuspended in serum-free medium. Equal volumes of 0.4% trypan blue and cell suspension were mixed and cells were counted using a hemacytometer. This assay was performed three times in triplicate.

Results

Inhibiting the m6A writer METTL3 using STM2457 (FIG. 8B) decreases the cell proliferation of UF cells as shown in FIG. 9.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.