Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Whole salivary gland generation and transplantation offer potential therapies for salivary gland dysfunction. However, the specific lineage required to engineer complete salivary glands has remained elusive. In this study, we identify the Foxa2 lineage as a critical lineage for salivary gland development through conditional blastocyst complementation (CBC). Foxa2 lineage marking begins at the boundary between the endodermal and ectodermal regions of the oral epithelium before the formation of the primordial salivary gland, thereby labeling the entire gland. Ablation of Fgfr2 within the Foxa2 lineage in mice leads to salivary gland agenesis. We reversed this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts, resulting in mice that survived to adulthood with salivary glands of normal size, comparable to those of their littermate controls. These findings demonstrate that CBC-based salivary gland regeneration serves as a foundational experimental approach for future advanced cell-based therapies.

Ephrin Forward Signaling Controls Interspecies Cell Competition in Pluripotent Stem Cells.

In the animal kingdom, evolutionarily conserved mechanisms known as cell competition eliminate unfit cells during development. Interestingly, cell competition also leads to apoptosis of donor cells upon direct contact with host cells from a different species during interspecies chimera formation. The mechanisms underlying how host animal cells recognize and transmit cell death signals to adjacent xenogeneic human cells remain incompletely understood. In this study, we developed an interspecies cell contact reporter system to dissect the mechanisms underlying competitive interactions between mouse and human pluripotent stem cells (PSCs). Through single-cell RNA-seq analyses, we discovered that Ephrin A ligands in mouse cells play a crucial role in signaling cell death to adjacent human cells that express EPHA receptors during interspecies PSC co-culture. We also demonstrated that blocking the Ephrin A-EPHA receptor interaction pharmacologically, and inhibiting Ephrin forward signaling genetically in the mouse cells, enhances the survival of human PSCs and promotes chimera formation both in vitro and in vivo . Our findings elucidate key mechanisms of interspecies PSC competition during early embryogenesis and open new avenues for generating humanized tissues or organs in animals, potentially revolutionizing regenerative medicine.

Statistical optimization of environmental factors to produce the cytotoxic enniatins H, I and MK1688 against human multidrug resistance cancer cell lines.

The environmental conditions were optimized to produce the enniatin H, I, and MK1688 by Fusarium strain on cereal grain exhibiting anti-carcinogenic potential against MES-SA (human uterine sarcoma cell line), HCT15 (human colorectal carcinoma cancer cell line), and their multidrug resistance sublines. From the statistical optimization by response surface methodology, the optimal condition of independent variables affecting the response variables were 20.85 °C (temperature), 46.85% (w/w, initial moisture content), and 18.42 days (growth time) for ENN H; 23.31 °C, 44.15% (w/w) and 17.23 days for ENN I; 23.08 °C, 43.97% (w/w) and 17.06 days for ENN MK1688. In case of cytotoxic effects, ENNs significantly suppressed growth of cancer cell lines without multidrug resistance, and ENN I inhibited growth of cancer cell lines most strongly. These data will provide valuable point to produce the cyclic hexadepsipeptide exhibiting anti-carcinogenic potential from Fusarium strains.

Comparative Evaluation of Egg Quality in Response to Temperature Variability: From Farm to Table Exposure Scenarios.

This study aimed to develop appropriate temperature management practices and provide scientific evidence to support the development of sell-by-date guidance for eggs. Washed and unwashed eggs were subjected to storage under six different scenarios, and both types of eggs were stored at temperatures up to 35°C to evaluate the sell-by-date. Despite temperature fluctuations or continuous storage at 30°C for 5 days, subsequent storage at 10°C resulted in significantly higher Haugh unit and yolk index on day 15. These results indicate that refrigerating eggs from retail sales until consumption is effective for egg quality management, despite the exposure of up to 35°C during distribution. In terms of sell-by-date evaluation, washed eggs retained class B quality for an additional 37 days beyond the recommended sell-by-date at 15°C, which is above the regulated storage temperature. However, unwashed eggs maintained class B quality for approximately 20 days at 30°C-35°C, emphasizing the need for sell-by-date guidelines for unwashed eggs. This study is the first to provide appropriate egg-handling practices based on the actual distribution environment in Korea.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Various patients suffer from dry mouth due to salivary gland dysfunction. Whole salivary gland generation and transplantation is a potential therapy to resolve this issue. However, the lineage permissible to design the entire salivary gland generation has been enigmatic. Here, we discovered Foxa2 as a lineage critical for generating a salivary gland via conditional blastocyst complementation (CBC). Foxa2 linage, but not Shh nor Pitx2, initiated to label between the boundary region of the endodermal and the ectodermal oral mucosa before primordial salivary gland formation, resulting in marking the entire salivary gland. The salivary gland was agenesis by depleting Fgfr2 under the Foxa2 lineage in the mice. We rescued this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts. Those mice survived until adulthood with normal salivary glands compatible in size compared with littermate controls. These results indicated that CBC-based salivary gland generation is promising for next-generation cell-based therapy.

Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes the generation of the whole lung.

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.

An immunostimulatory glycolipid that blocks SARS-CoV-2, RSV, and influenza infections in vivo.

Prophylactic vaccines for SARS-CoV-2 have lowered the incidence of severe COVID-19, but emergence of viral variants that are antigenically distinct from the vaccine strains are of concern and additional, broadly acting preventive approaches are desirable. Here, we report on a glycolipid termed 7DW8-5 that exploits the host innate immune system to enable rapid control of viral infections in vivo. This glycolipid binds to CD1d on antigen-presenting cells and thereby stimulates NKT cells to release a cascade of cytokines and chemokines. The intranasal administration of 7DW8-5 prior to virus exposure significantly blocked infection by three different authentic variants of SARS-CoV-2, as well as by respiratory syncytial virus and influenza virus, in mice or hamsters. We also found that this protective antiviral effect is both host-directed and mechanism-specific, requiring both the CD1d molecule and interferon-[Formula: see text]. A chemical compound like 7DW8-5 that is easy to administer and cheap to manufacture may be useful not only in slowing the spread of COVID-19 but also in responding to future pandemics long before vaccines or drugs are developed.

Construction of a bioactive copper-based metal organic framework-embedded dual-crosslinked alginate hydrogel for antimicrobial applications.

Metal-organic frameworks (MOFs) containing bioactive metals have the potential to exhibit antimicrobial activity by releasing metal ions or ligands through the cleavage of metal-ligand bonds. Recently, copper-based MOFs (Cu-MOFs) with sustained release capability, porosity, and structural flexibility have shown promising antimicrobial properties. However, for clinical use, the controlled release of Cu2+ over an extended time period is crucial to prevent toxicity. In this study, we developed an alginate-based antimicrobial scaffold and encapsulated MOFs within a dual-crosslinked alginate polymer network. We synthesized Cu-MOFs containing glutarate (Glu) and 4,4'-azopyridine (AZPY) (Cu(AZPY)-MOF) and encapsulated them in an alginate-based hydrogel through a combination of visible light-induced photo and calcium ion-induced chemical crosslinking processes. We confirmed Cu(AZPY)-MOF synthesis using scanning electron microscopy, transmission electron microscopy, powder X-ray diffraction, and thermogravimetric analysis. This antimicrobial hydrogel demonstrated excellent antibacterial and antifungal properties against two bacterial strains (MRSA and S. mutans, with >99.9 % antibacterial rate) and one fungal strain (C. albicans, with >78.7 % antifungal rate) as well as negligible cytotoxicity towards mouse embryonic fibroblasts, making it a promising candidate for various tissue engineering applications in biomedical fields.

Simulation of contamination and elimination of Escherichia coli, Listeria monocytogenes, and Murine norovirus 1 (MNV-1) from the washing process when handling of potatoes.

Root vegetables, which are in close contact with soil, are particularly vulnerable to soil contamination or decay as they can be contaminated from multiple sources, including primary production and processing. This study investigated effective washing conditions to reduce the microbial contamination of potatoes by using soaking and shaking in the washing process. The reduction of Escherichia coli, Listeria monocytogenes, and Murine norovirus 1 (MNV-1) in four washing processes (soaking only, shaking only, combined soaking-shaking I, and combined soaking-shaking I-shaking II) were compared. The numbers of E. coli and L. monocytogenes decreased by 0.55 and 0.49 log CFU/g after shaking only, 1.96 and 1.80 log CFU/g after soaking, 2.07 and 1.67 log CFU/g after soaking-shaking I, and 2.42 and 1.90 log CFU/g after soaking-shaking I-shaking II, respectively. The combined process reduced the microbial contamination more efficiently than shaking only. The reduction of E. coli in the washing process was higher than that of L. monocytogenes by approximately 0.5 logs. MNV-1 showed a reduction in the soaking and shaking steps by 1.34 and 1.98 log GC/100 g, with no significant reduction observed after the combination process. A combined process of soaking-shaking I-shaking II was effective to eliminate E. coli, L. monocytogenes, and MNV-1 from potatoes during the handling and washing process.

Towards human organ generation using interspecies blastocyst complementation: Challenges and perspectives for therapy.

Millions of people suffer from end-stage refractory diseases. The ideal treatment option for terminally ill patients is organ transplantation. However, donor organs are in absolute shortage, and sadly, most patients die while waiting for a donor organ. To date, no technology has achieved long-term sustainable patient-derived organ generation. In this regard, emerging technologies of chimeric human organ production via blastocyst complementation (BC) holds great promise. To take human organ generation via BC and transplantation to the next step, we reviewed current emerging organ generation technologies and the associated efficiency of chimera formation in human cells from the standpoint of developmental biology.

Injectable hyaluronic acid hydrogel encapsulated with Si-based NiO nanoflower by visible light cross-linking: Its antibacterial applications.

Bacterial infections have become a severe threat to human health and antibiotics have been developed to treat them. However, extensive use of antibiotics has led to multidrug-resistant bacteria and reduction of their therapeutic effects. An efficient solution may be localized application of antibiotics using a drug delivery system. For clinical application, they need to be biodegradable and should offer a prolonged antibacterial effect. In this study, a new injectable and visible-light-crosslinked hyaluronic acid (HA) hydrogel loaded with silicon (Si)-based nickel oxide (NiO) nanoflowers (Si@NiO) as an antibacterial scaffold was developed. Si@NiO nanoflowers were synthesized using chemical bath deposition before encapsulating them in the HA hydrogel under a mild visible-light-crosslinking conditions to generate a Si@NiO-hydrogel. Si@NiO synthesis was confirmed using scanning electron microscopy, transmission electron microscopy, and powder X-ray diffraction. As-prepared Si@NiO-hydrogel exhibited enhanced mechanical properties compared to a control bare hydrogel sample. Moreover, Si@NiO-hydrogel exhibits excellent antibacterial properties against three bacterial strains (P. aeruginosa, K. pneumoniae, and methicillin-resistant Staphylococcus aureus (>99.9% bactericidal rate)) and negligible cytotoxicity toward mouse embryonic fibroblasts. Therefore, Si@NiO-hydrogel has the potential for use in tissue engineering and biomedical applications owing to its injectability, visible-light crosslink ability, degradability, biosafety, and superior antibacterial property.

High Device Performances and Noise Characteristics of AlGaN/GaN HEMTs Using In Situ SiCN and SiN Cap Layer.

We fabricated and characterized AlGaN/GaN high-electron mobility transistors (HEMTs) with a nano-sized in situ cap layer (one is a silicon carbon nitride (SiCN) layer, and the other is a silicon nitride (SiN) layer) comparing to the conventional device without an in situ cap layer. The pulse characteristics and noise behaviors for two devices with in situ cap layers are much superior to those of the reference device without a cap layer, which means that the in situ cap layer effectively passivates the AlGaN surface. On the other hand, the device with an in situ SiCN cap layer showed the excellent device characteristics and noise performances compared to the other devices because of the reduced positive ionic charges and enhanced surface morphology caused by carbon (C) surfactant atoms during the growth of the SiCN cap layer. These results indicate that the AlGaN/GaN HEMT with the in situ SiCN cap layer is very promising for the next high-power device by replacing the conventional HEMT.

All-in-one nanosponge with pluronic shell for synergistic anticancer therapy through effectively overcoming multidrug resistance in cancer.

Overexpression of P-glycoprotein (P-gp) on cancer cells is a major hurdle to effectively treat tumors with multidrug resistance (MDR). The current study aimed to explore anticancer drug and P-gp inhibitor delivery as a promising strategy to efficiently treat colorectal cancer with MDR. To this end, a multidrug-loaded all-in-one nanosponge (ANS) was developed to simultaneously deliver doxorubicin (DOX), paclitaxel (PTX), and the P-gp inhibitor tetrandrine (TET), referred to as DOX/PTX/TET@ANS, without chemical conjugation. ANS with high loading content and efficiency facilitated a pH-dependent and controlled release with different profiles. Compared to free drugs and DOX/PTX@ANS, DOX/PTX/TET@ANS exhibited more effective anticancer effects on P-gp-overexpressing colorectal cancer cells and solid tumor mouse xenografts, without major toxicity. Notably, ANS composed of pluronic shell induced in vitro P-gp inhibition compared to TET, implying a synergistic anticancer effect. These findings suggest that ANS can encapsulate multiple drugs to efficiently deliver chemotherapy, particularly in MDR tumors.

Enhanced Transport and Permeation of a Polymeric Nanocarrier across the Retina by Mixing with ATP upon Intravitreal Injection.

The outer part of the retina pigment epithelium (RPE) in the retina is the main site of neovascularization associated with retinal diseases. However, various obstacles interrupt the delivery of medicines across the RPE, mainly due to the well-developed tight junctions in the RPE. Currently, there is no practical formulation to overcome this issue. In this study, we demonstrated that simple mixing with adenosine tetraphosphate (ATP) has the potential to greatly enhance the transport and permeation of a polymeric nanocarrier across the retina via intravitreal administration. Chitosan-functionalized, pluronic-based nanocarrier (NC), which can deliver various biomolecules efficiently, was used as a polymeric nanocarrier. Mixing with ATP facilitated the diffusion of the nanocarrier in the vitreous humor by reducing the electrostatic interaction between NC and negatively charged glycosaminoglycans (GAGs) in the vitreous humor. Mixing with ATP also allowed the penetration of NC across the whole retina, and it resulted in a great increase (approximately nine times) in the transport of NC across the retina, as well as spreading it throughout the whole retina upon intravitreal administration in a mouse model. This enhanced permeation across the retina was specific to ATP but not to GTP, suggesting the possibility of P2Y receptor-mediated tight junction disruption by ATP.

Characteristics of GaN-Based Nanowire Gate-All-Around (GAA) Transistors.

We investigate the DC, C-V, and pulse performances in GaN-based nanowire gate-all-around (GAA) transistors with two kinds of geometry: one is AlGaN/GaN heterostructure with two dimensional electron gas (2DEG) channel and the other is only GaN layer without 2DEG channel. From I-V and C-V curves, the fabricated GaN nanowire GAA transistor with AlGaN layer clearly exhibits normally-on operation with negative threshold voltage (Vth) due to the existence of 2DEG channel on the trapezoidal shaped GaN nanowire. On the other hand, the GaN nanowire GAA transistor without AlGaN layer presents a positive Vth (normally-off operation) due to the absent of 2DEG channel on the triangle shaped GaN nanowire. However, both devices show the similar temperaturedependent I-V characteristics due to the combination of bulk channel and surface channel in GaN nanowire GAA channel are mostly contributed, rather than the 2DEG channel. GaN-based nanowire GAA transistors demonstrate to almost negligible current collapse phenomenon due to the perfect GAA gate structure in GaN nanowire. The proposed GaN-based nanowire GAA transistors are very promising candidate for both high power device and nano-electronics application.

Targeted delivery of anti-inflammatory cytokine by nanocarrier reduces atherosclerosis in Apo E-/- mice.

Unresolved inflammation is a hallmark of many deadly diseases including atherosclerosis, a silent pathological condition behind majority of cardiovascular diseases. Yet, anti-inflammatory drugs are not clinically used in the treatment of patients with atherosclerosis. The currently approved treatment regimen against atherosclerosis is mainly focused on lowering the cholesterol/lipid levels in blood and has little to do with controlling inflammation, the underlying cause. Recent preclinical and clinical data suggest that effective alleviation of inflammation in the atherosclerosis plaque could reduce the risk of cardiovascular disease. In this work, we have encapsulated interleukin-10 (IL10), a multipotent anti-inflammatory cytokine into cRGD conjugated pluronic based nano-carriers (NC) for targeted delivery to atherosclerotic plaques. The NC could encapsulate the therapeutic protein with a high loading efficiency in a mild condition and showed sustained release capabilities. The efficacy of cytokine encapsulated NC was analyzed in vitro using the lipopolysaccharide stimulated macrophage cells and in vivo using an established apolipoprotein E-knockout (ApoE-/-) C57BL/6 mouse model. Compared to free IL10, intravenous administration of NC encapsulated IL10 resulted in vastly improved pharmacokinetic profile and profoundly high accumulation of the cytokine in the atherosclerosis lesions. IL10 delivered by NC was bioactive and reduced the production of pro-inflammatory cytokine IL-1ß in the lesion and led to significant regression in the plaque size. These results signify the prospect of nanoparticle based cytokine delivery for preventing atherosclerotic through inflammation modulation in near future.

Significantly enhanced recovery of acute liver failure by liver targeted delivery of stem cells via heparin functionalization.

Acute liver failure (ALF) occurs by insufficient detoxification of toxic materials in the liver, generating excess reactive oxygen species (ROS). Mesenchymal stem cell (MSC) therapy can be a promising approach for the treatment of liver diseases including ALF by anti-inflammatory activity and secretion of cytokines associated with tissue regeneration. However, the efficacy of MSC therapy is generally poor, mainly due to a low survival and engraftment of administered cells. In this study, we demonstrated that the enhanced delivery of human adipose-derived stem cells (hADSCs) to the damaged liver by the coating of lipid-conjugated heparin could result in significantly improved recovery from ALF in a mouse model. First, the therapeutic effect of secretomes of hADSCs on acetaminophen (APAP)-induced hepatic cell damage was confirmed regardless of the coating of lipid-conjugated heparin on hADSCs in vitro. Then, the therapeutic effects of lipid-conjugated heparin coated hADSCs (Lip-Hep/hADSC group) were analyzed compared to hADSCs themselves (hADSC group) using an APAP-induced ALF model in vivo. Intravenous administration of hADSCs could lower the elevated serum levels of aspartate transaminase (AST) and alanine transaminase (ALT), but Lip-Hep/hADSC group showed faster decrease in serum levels of AST and ALT to the normal values compared to hADSC group. Enhanced delivery and longer retention of hADSCs in the damage liver by the coating of lipid-conjugated heparin were confirmed by optical imaging of isolated organs using labeled cells and immunofluorescence staining of liver tissue sections against human nuclei. A significantly increased level of human hepatic growth factor (hHGF), a representative secretome from hADSC, significantly reduced levels of macrophage and CYP2E1, implying alleviated inflammatory response, were detected by immunofluorescence staining from Lip-Hep/hADSC group compared to hADSC group. These results well coincided with the improved recovery of the damaged liver from Lip-Hep/hADSC group than hADSC group in histological analysis. Thus, the coating of lipid-conjugated heparin on hADSCs has a great potential to improve the therapeutic effect of cells on the liver injury.

Photoelectrochemical Gas-Electrolyte-Solid Phase Boundary for Hydrogen Production From Water Vapor.

Hydrogen production from humidity in the ambient air reduces the maintenance costs for sustainable solar-driven water splitting. We report a gas-diffusion porous photoelectrode consisting of tungsten trioxide (WO3) nanoparticles coated with a proton-conducting polymer electrolyte thin film for visible-light-driven photoelectrochemical water vapor splitting. The gas-electrolyte-solid triple phase boundary enhanced not only the incident photon-to-current conversion efficiency (IPCE) of the WO3 photoanode but also the Faraday efficiency (FE) of oxygen evolution in the gas-phase water oxidation process. The IPCE was 7.5% at an applied voltage of 1.2 V under 453 nm blue light irradiation. The FE of hydrogen evolution in the proton exchange membrane photoelectrochemical cell was close to 100%, and the produced hydrogen was separated from the photoanode reaction by the membrane. A comparison of the gas-phase photoelectrochemical reaction with that in liquid-phase aqueous media confirmed the importance of the triple phase boundary for realizing water vapor splitting.

An injectable and physical levan-based hydrogel as a dermal filler for soft tissue augmentation.

The use of injectable materials as a biofiller for soft tissue augmentation has been increasing worldwide. Levan is a biocompatible and inexpensive polysaccharide with great potential in biomaterial applications, but it has not been extensively studied. In this study, we evaluated the potential of levan as a new material for dermal fillers and prepared an injectable and physical levan-based hydrogel by combining levan with Pluronic and carboxymethyl cellulose (CMC). A sol state was prepared by mixing the polymers in a specific ratio at 4 °C for 2 days and the hydrogel was formed by increasing the temperature to 37 °C. The elastic modulus of the levan hydrogel was higher than that of a hyaluronic acid (HA)-based hydrogel. The SEM images of the levan hydrogel showed an interconnected porous structure, similar to the HA hydrogel. Levan showed non-cytotoxicity, enhanced cell proliferation, and higher amount of collagen synthesis in human dermal fibroblast cells compared to HA. The injected levan hydrogel was biocompatible and stable over 2 weeks in vivo, longer than the Pluronic F127 hydrogel or HA hydrogel. Also, the levan hydrogel showed a higher amount of collagen production than the HA hydrogel in vivo. More importantly, the levan hydrogel showed enhanced anti-wrinkle efficacy compared to the HA hydrogel in a wrinkle model mouse. Thus, the levan hydrogel with injectability, biocompatibility, and an anti-wrinkle effect has high potential as an alternative to existing commercial dermal fillers.

A novel alendronate functionalized nanoprobe for simple colorimetric detection of cancer-associated hypercalcemia.

The calcium (Ca2+) ion concentration in the blood serum is tightly regulated, and any abnormalities in the level of serum calcium ions are associated with many potentially dangerous diseases. Thus, monitoring of the Ca2+ ion concentration in the blood serum is of fundamental importance. Gold nanoparticle (GNP)-based colorimetric biosensors have enormous potential in clinical diagnostic applications due to their simplicity, versatility, and unique optical properties. In this study, we have developed an alendronate functionalized gold nanoparticle (GNP-ALD) system for the measurement of Ca2+ ion concentration in biological samples. The GNP-ALD system showed higher sensitivity towards the Ca2+ ion compared to adenosine diphosphate (ADP) or adenosine triphosphate (ATP). The strong interaction between the Ca2+ ion and ALD at the GNP/solution interface resulted in significant aggregation of the ALD conjugated GNPs, and induced a color change of the solution from red to blue, which could be visually observed with the naked eye. The interaction between the Ca2+ ion and GNP-ALD was characterized by UV-visible spectroscopy, transmission electron microscopy (TEM) imaging, and dynamic light scattering (DLS) analysis. Under the optimized conditions, the lower limit of Ca2+ ion detection using this method was found to be 25 µM and a linear response range from 25 µM to 300 µM Ca2+ ions was obtained with excellent discrimination against other metal ions. The GNP-ALD nanoprobe could successfully determine the ionized Ca2+ concentration in various serum samples and the results were validated using a commercial calcium assay kit. Moreover, as a practical application, we demonstrated the utility of this nanoprobe for the detection of cancer-associated hypercalcemia in a mouse model.