Enhancing the Bioavailability and Bioactivity of Curcumin for Disease Prevention and Treatment.

Curcumin, a natural polyphenolic component from Curcuma longa roots, is the main bioactive component of turmeric spice and has gained increasing interest due to its proposed anti-cancer, anti-obesity, anti-inflammatory, antioxidant, and lipid-lowering effects, in addition to its thermogenic capacity. While intake from dietary sources such as curry may be sufficient to affect the intestinal microbiome and thus may act indirectly, intact curcumin in the body may be too low (<1 microM) and not sufficient to affect signaling and gene expression, as observed in vitro with cultured cells (10-20 microM). Several strategies can be envisioned to increase curcumin levels in the body, such as decreasing its metabolism or increasing absorption through the formation of nanoparticles. However, since high curcumin levels could also lead to undesired regulatory effects on cellular signaling and gene expression, such studies may need to be carefully monitored. Here, we review the bioavailability of curcumin and to what extent increasing curcumin levels using nanoformulations may increase the bioavailability and bioactivity of curcumin and its metabolites. This enhancement could potentially amplify the disease-preventing effects of curcumin, often by leveraging its robust antioxidant properties.

A Portable, Encapsulated Microbial Whole-Cell Biosensing System for the Detection of Bioavailable Copper (II) in Soil.

A portable, field deployable whole-cell biosensor was developed that can withstand the complex matrices of soil and requires minimal to no sample preparation to monitor bioavailable concentrations of the essential micronutrient copper (II). Conventional measurement of micronutrients is often complex, laboratory-based, and not suitable for monitoring their bioavailable concentration. To address this need, we developed a fluorescence based microbial whole-cell biosensing (MWCB) system encoding for a Cu2+-responsive protein capable of generating a signal upon binding to Cu2+. The sensing-reporting protein was designed by performing circular permutation on the green fluorescent protein (GFP) followed by insertion of a Cu2+ binding motif into the structure of GFP. The design included insertion of several binding motifs and creating plasmids that encoded the corresponding sensing proteins. The signal generated by the sensing-reporting protein is directly proportional to the concentration of Cu2+ in the sample. Evaluation of the resulting biosensing systems carrying these plasmids was performed prior to selection of the optimal fluorescence emitting Cu2+-binding protein. The resulting optimized biosensing system was encapsulated in polyacrylate-alginate beads and embedded in soil for detection of the analyte. Once exposed to the soil, the beads were interrogated to measure the fluorescence signal emitted by the sensing-reporting protein using a portable imaging device. The biosensor was optimized for detection of Cu2+ in terms of selectivity, sensitivity, matrix effects, detection limits, and reproducibility in both liquid and soil matrices. The limit of detection (LoD) of the optimized encapsulated biosensor was calculated as 0.27 mg/L and 1.26 mg/kg of Cu2+ for Cu2+ in solution and soil, respectively. Validation of the portable imaging tools as a potential biosensing device in the field was performed.

Point-of-Care Molecular Test for the Detection of 14 High-Risk Genotypes of Human Papillomavirus in a Single Tube.

Cervical cancers constitute a large disease burden in developing countries, with the human papillomavirus (HPV) being responsible for most cervical lesions. Many regions in low-resource countries lack adequate access to sensitive point-of-care (POC) screening tools, preventing timely diagnosis and treatment. To reduce screening barriers, we developed a POC HPV molecular test that detects 14 high-risk HPV types in 30 min in a single assay. We introduced innovations to the underlying amplification (recombinase polymerase amplification) and detection methodologies such as improved probe design, reagent lyophilization, and pipette-less processing to increase sensitivity while enabling minimally trained personnel to conduct reproducible testing. Based on 198 clinically derived samples, we demonstrated a sensitivity of 93% and a specificity of 73% compared to an FDA-approved polymerase chain reaction-based clinical method. Our modified pipette-less simplified assay had a sensitivity of 96% and a specificity of 83%. The application of our assay is intended as a near-patient screening tool with further evaluation by a clinician for confirmation.

Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19).

Rapid on-site diagnosis of emerging pathogens is key for early identification of infected individuals and for prevention of further spreading in a population. Currently available molecular diagnostic tests are instrument-based whereas rapid antibody and antigen tests are often not sufficiently sensitive for detection in pre-symptomatic subjects. There is a need for rapid point of care molecular screening tests that can be easily adapted to emerging pathogens and are selective, sensitive, reliable in different settings around the world. We have developed a simple, rapid (<30 â€‹min), and inexpensive test for SARS-CoV-2 that is based on combination of isothermal reverse transcription recombinase polymerase amplification (RT-RPA) using modified primers and visual detection with paper-based microfluidics. Our test (CoRapID) is specific for SARS-CoV-2 (alpha to omicron variants) and does not detect other coronaviruses and pathogens by in silico and in vitro analysis. A two-step test protocol was developed with stable lyophilized reagents that reduces handling by using portable and disposable components (droppers, microapplicators/swabs, paper-strips). After optimization of assay components and conditions, we have achieved a limit of detection (LoD) of 1 copy/reaction by adding a blocking primer to the lateral flow assay. Using a set of 138 clinical samples, a sensitivity of 88.1% (P â€‹< â€‹0.05, CI: 78.2-93.8%) and specificity of 93.9% (P â€‹< â€‹0.05, CI: 85.4-97.6%) was determined. The lack of need for instrumentation for our CoRapID makes it an ideal on-site primary screening tool for local hospitals, doctors' offices, senior homes, workplaces, and in remote settings around the world that often do not have access to clinical laboratories.

Engineered biosensors for the quorum sensing molecule 3,5-dimethyl-pyrazine-2-ol (DPO) reveal its presence in humans, animals, and bacterial species beyond Vibrio cholerae.

A biosensor was engineered to enable the study of the novel quorum sensing molecule (QSM), 3,5-dimethylpyrazin-2-ol (DPO), employed by Vibrio cholerae to regulate biofilm formation and virulence factor production. Investigations into bacterial quorum sensing (QS), a form of communication based on the production and detection of QSMs to coordinate gene expression in a population dependent manner, offer a unique window to study the molecular underpinnings of microbial behavior and host interactions. Herein, we report the construction of an engineered microbial whole-cell bioluminescent biosensing system that incorporates the recognition of the VqmA regulatory protein of Vibrio cholerae with the bioluminescent reporting signal of luciferase for the selective, sensitive, stable, and reproducible detection of DPO in a variety of samples. Importantly, using our newly developed biosensor our studies demonstrate the detection of DPO in rodent and human samples. Employing our developed biosensor should help enable elucidation of microbial behavior at the molecular level and its impact in health and disease.

The difference in the cellular uptake of tocopherol and tocotrienol is influenced by their affinities to albumin.

Vitamin E is classified into tocopherol (Toc) and tocotrienol (T3) based on its side chains. T3 generally has higher cellular uptake than Toc, though the responsible mechanism remains unclear. To elucidate this mechanism, we hypothesized and investigated whether serum albumin is a factor that induces such a difference in the cellular uptake of Toc and T3. Adding bovine serum albumin (BSA) to serum-depleted media increased the cellular uptake of T3 and decreased that of Toc, with varying degrees among α-, ß-, γ-, and δ-analogs. Such enhanced uptake of α-T3 was not observed when cells were incubated under low temperature (the uptake of α-Toc was also reduced), suggesting that Toc and T3 bind to albumin to form a complex that results in differential cellular uptake of vitamin E. Fluorescence quenching study confirmed that vitamin E certainly bound to BSA, and that T3 showed a higher affinity than Toc. Molecular docking further indicated that the differential binding energy of Toc or T3 to BSA is due to the Van der Waals interactions via their side chain. Overall, these results suggested that the affinity of Toc and T3 to albumin differs due to their side chains, causing the difference in their albumin-mediated cellular uptake. Our results give a better mechanistic insight into the physiological action of vitamin E.

Bioactive dietary components-Anti-obesity effects related to energy metabolism and inflammation.

Obesity is the result of the long-term energy imbalance between the excess calories consumed and the few calories expended. Reducing the intake of energy dense foods (fats, sugars), and strategies such as fasting and caloric restriction can promote body weight loss. Not only energy in terms of calories, but also the specific composition of the diet can affect the way the food is absorbed and how its energy is stored, used or dissipated. Recent research has shown that bioactive components of food, such as polyphenols and vitamins, can influence obesity and its pathologic complications such as insulin resistance, inflammation and metabolic syndrome. Individual micronutrients can influence lipid turnover but for long-term effects on weight stability, dietary patterns containing several micronutrients may be required. At the molecular level, these molecules modulate signaling and the expression of genes that are involved in the regulation of energy intake, lipid metabolism, adipogenesis into white, beige and brown adipose tissue, thermogenesis, lipotoxicity, adipo/cytokine synthesis, and inflammation. Higher concentrations of these molecules can be reached in the intestine, where they can modulate the composition and action of the microbiome. In this review, the molecular mechanisms by which bioactive compounds and vitamins modulate energy metabolism, inflammation and obesity are discussed.

Modulation of CD36-mediated lipid accumulation and senescence by vitamin E analogs in monocytes and macrophages.

The CD36/FAT scavenger receptor/fatty acids transporter regulates cellular lipid accumulation important for inflammation, atherosclerosis, lipotoxicity, and initiation of cellular senescence. Here we compared the regulatory effects of the vitamin E analogs alpha-tocopherol (αT), alpha-tocopheryl phosphate (αTP), and αTP/ßCD (a nanocarrier complex between αTP and ß-cyclodextrin [ßCD]) and investigated their regulatory effects on lipid accumulation, phagocytosis, and senescence in THP-1 monocytes and macrophages. Both, αTP and αTP/ßCD inhibited CD36 surface exposition stronger than αT leading to more pronounced CD36-mediated events such as inhibition of DiI-labeled oxLDL uptake, phagocytosis of fluorescent Staphylococcus aureus bioparticles, and cell proliferation. When compared to ßCD, the complex of αTP/ßCD extracted cholesterol from cellular membranes with higher efficiency and was associated with the delivery of αTP to the cells. Interestingly, both, αTP and more so αTP/ßCD inhibited lysosomal senescence-associated beta-galactosidase (SA-ß-gal) activity and increased lysosomal pH, suggesting CD36-mediated uptake into the endo-lysosomal phagocytic compartment. Accordingly, the observed pH increase was more pronounced with αTP/ßCD in macrophages whereas no significant increase occurred with αT, alpha-tocopheryl acetate (αTA) or ßCD. In contrast to αT and αTA, the αTP molecule is di-anionic at neutral pH, but upon moving into the acidic endo-lysosomal compartment becomes protonated and thus is acting as a base. Moreover, it is expected to be retained in lysosomes since it still carries one negative charge, similar to lysosomotropic drugs. Thus, treatment with αTP or αTP/ßCD and/or inhibition of conversion of αTP to αT as it occurs in aged cells may counteract CD36-mediated overlapping inflammatory, senescent, and atherosclerotic events.

Oxidized LDLs as Signaling Molecules.

Levels of oxidized low-density lipoproteins (oxLDLs) are usually low in vivo but can increase whenever the balance between formation and scavenging of free radicals is impaired. Under normal conditions, uptake and degradation represent the physiological cellular response to oxLDL exposure. The uptake of oxLDLs is mediated by cell surface scavenger receptors that may also act as signaling molecules. Under conditions of atherosclerosis, monocytes/macrophages and vascular smooth muscle cells highly exposed to oxLDLs tend to convert to foam cells due to the intracellular accumulation of lipids. Moreover, the atherogenic process is accelerated by the increased expression of the scavenger receptors CD36, SR-BI, LOX-1, and SRA in response to high levels of oxLDL and oxidized lipids. In some respects, the effects of oxLDLs, involving cell proliferation, inflammation, apoptosis, adhesion, migration, senescence, and gene expression, can be seen as an adaptive response to the rise of free radicals in the vascular system. Unlike highly reactive radicals, circulating oxLDLs may signal to cells at more distant sites and possibly trigger a systemic antioxidant defense, thus elevating the role of oxLDLs to that of signaling molecules with physiological relevance.

Microbial whole-cell biosensors: Current applications, challenges, and future perspectives.

Microbial Whole-Cell Biosensors (MWCBs) have seen rapid development with the arrival of 21st century biological and technological capabilities. They consist of microbial species which produce, or limit the production of, a reporter protein in the presence of a target analyte. The quantifiable signal from the reporter protein can be used to determine the bioavailable levels of the target analyte in a variety of sample types at a significantly lower cost than most widely used and well-established analytical instrumentation. Furthermore, the versatile and robust nature of MWCBs shows great potential for their use in otherwise unavailable settings and environments. While MWCBs have been developed for use in biomedical, environmental, and agricultural monitoring, they still face various challenges before they can transition from the laboratory into industrialized settings like their enzyme-based counterparts. In this comprehensive and critical review, we describe the underlying working principles of MWCBs, highlight developments for their use in a variety of fields, detail challenges and current efforts to address them, and discuss exciting implementations of MWCBs helping redefine what is thought to be possible with this expeditiously evolving technology.

Reduction of senescence-associated beta-galactosidase activity by vitamin E in human fibroblasts depends on subjects' age and cell passage number.

Cell senescence is due to the permanent cell cycle arrest that occurs as a result of the inherent limited replicative capacity toward the Hayflick limit (replicative senescence), or in response to various stressors (stress-induced premature senescence, SIPS). With the acquisition of the senescence-associated secretory phenotype (SASP), cells release several molecules (cytokines, proteases, lipids), and express the senescence-associated beta-galactosidase (SA-ß-Gal). Here we tested whether vitamin E affects SA-ß-Gal in an in vitro model of cell ageing. Skin fibroblasts from human subjects of different age (1, 13, 29, 59, and 88 years old) were cultured until they reached replicative senescence. At different passages (Passages 2, 9, 13, and 16), these cells were treated with vitamin E for 24 hr. Vitamin E reduced SA-ß-Gal in all cells at passage 16, but at earlier passage numbers it reduced SA-ß-Gal only in cells isolated from the oldest subjects. Therefore, short time treatment with vitamin E decreases SA-ß-Gal in cells both from young and old subjects when reaching replicative senescence; but in cells isolated from older subjects, a decrease in SA-ß-Gal by vitamin E occurs also at earlier passage numbers. The possible role of downregulation of CD36 by vitamin E, a scavenger receptor essential for initiation of senescence and SASP, is discussed.

The differential cellular uptake of curcuminoids in vitro depends dominantly on albumin interaction.

BACKGROUND: Curcuminoids, mainly present in the plant rhizomes of turmeric (Curcuma longa), consist of mainly three forms (curcumin (CUR), bisdemethoxycurcumin (BDMC) and demethoxycurcumin (DMC)). It has been reported that different forms of curcuminoids possess different biological activities. However, the mechanisms associated with these differences are not well-understood. Recently, our laboratory found differences in the cellular uptake of these curcuminoids. Therefore, it has been inferred that these differences contribute to the different biological activities. PURPOSE: In this study, we investigated the mechanisms of differential cellular uptake of these curcuminoids. METHOD: Based on our previous study, we hypothesized the differential cellular uptake is caused by (I) polarity, (II) transporters, (III) metabolism rate of curcuminoids and (IV) medium components. These four hypotheses were each investigated by (I) neutralizing the polarities of curcuminoids by encapsulation into poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs), (II) inhibition of polyphenol-related absorption transporters, (III) analysis of the cellular curcuminoids and their metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (IV) use of different mediums in cell study. RESULTS: The differential cellular uptake was not affected by (I-III). However, when investigating (IV), not only CUR but also BDMC and DMC were incorporated into cells when serum free media was used. Furthermore, when we used the serum free medium containing bovine serum albumin (BSA), only CUR was taken up but BDMC and DMC were not. Therefore, we identified that the differential cellular uptake of curcuminoids is caused by the medium components, especially BSA. Also, the fluorescence quenching study suggested that differential cellular uptake is due to the different interaction between BSA and each curcuminoid. CONCLUSION: The differential cellular uptake of curcuminoids was caused by the different interaction between curcuminoids and BSA. The results from this study might give clues on the mechanisms by which curcuminoids exhibit different physiological activities.

Vitamin E: Regulatory Role on Signal Transduction.

Vitamin E modulates signal transduction pathways by several molecular mechanisms. As a hydrophobic molecule located mainly in membranes it contributes together with other lipids to the physical and structural characteristics such as membrane stability, curvature, fluidity, and the organization into microdomains (lipid rafts). By acting as the main lipid-soluble antioxidant, it protects other lipids such as mono- and poly-unsaturated fatty acids (MUFA and PUFA, respectively) against chemical reactions with reactive oxygen and nitrogen species (ROS and RNS, respectively) and prevents membrane destabilization and cellular dysfunction. In cells, vitamin E affects signaling in redox-dependent and redox-independent molecular mechanisms by influencing the activity of enzymes and receptors involved in modulating specific signal transduction and gene expression pathways. By protecting and preventing depletion of MUFA and PUFA it indirectly enables regulatory effects that are mediated by the numerous lipid mediators derived from these lipids. In recent years, some vitamin E metabolites have been observed to affect signal transduction and gene expression and their relevance for the regulatory function of vitamin E is beginning to be elucidated. In particular, the modulation of the CD36/FAT scavenger receptor/fatty acids transporter by vitamin E may influence many cellular signaling pathways relevant for lipid homeostasis, inflammation, survival/apoptosis, angiogenesis, tumorigenesis, neurodegeneration, and senescence. Thus, vitamin E has an important role in modulating signal transduction and gene expression pathways relevant for its uptake, distribution, metabolism, and molecular action that when impaired affect physiological and patho-physiological cellular functions relevant for the prevention of a number of diseases. © 2018 IUBMB Life, 71(4):456-478, 2019.

Curcumin and piperine supplementation of obese mice under caloric restriction modulates body fat and interleukin-1ß.

BACKGROUND: Dietary bioactive compounds capable of improving metabolic profiles would be of great value, especially for overweight individuals undergoing a caloric restriction (CR) regimen. Curcumin (Cur), a possible anti-obesity compound, and piperine (Pip), a plausible enhancer of Cur's bioavailability and efficacy, may be candidate agents for controlling body fat, metabolism and low grade inflammation. METHODS: 47 eight-week-old male C57BL/6 mice were fed a high fat diet (HFD) for 23 weeks to induce obesity. Then, mice were divided into 5 groups. Group 1 continued on HFD ad libitum. The other 4 groups underwent CR (reduced 10% HFD intake for 10 weeks, 20% for 20 weeks) with Cur, Pip, Cur + Pip or none of these. Percent body fat, plasma inflammatory markers associated with obesity (interferon (IFN)-γ, interleukin (IL)-10, IL-12 p70, IL-1ß, IL-6 and KC/GRO), plasma Cur metabolites and liver telomere length were measured. RESULTS: Compared to the other groups, obese mice who underwent CR and received Cur + Pip in their diet lost more fat and had significantly lower IL-1ß and KC/GRO. Tandem mass spectrometry analysis of plasma from obese mice under CR showed no difference in Cur metabolite levels between groups supplemented with Cur alone or combined with Pip. However, plasma IL-1ß levels were inversely correlated with curcumin glucuronide. Minor modulation of telomere length were observed. CONCLUSIONS: It is plausible that supplementing the high fat diet of CR mice with Cur + Pip may increase loss of body fat and suppresses HFD induced inflammation. Combination of Cur and Pip has potential to enhance CR effects for the prevention of metabolic syndrome.

Water-Soluble Vitamin E-Tocopheryl Phosphate.

The hydrophobicity of vitamin E poses transport and metabolic challenges to regulate its bioavailability and to prevent its accumulation in lipid-rich tissues such as adipose tissue, brain, and liver. Water-soluble precursors of vitamin E (α-tocopherol, αT), such as its esters with acetate (αTA), succinate (αTS), or phosphate (αTP), have increased solubility in water and stability against reaction with free radicals, but they are rapidly converted during their uptake into the lipid-soluble vitamin E. Therefore, the bioavailability of these precursors as intact molecules is low; nevertheless, at least for αTS and αTP, the recent research has revealed unique regulatory effects on signal transduction and gene expression and the modulation of cellular events ranging from proliferation, survival/apoptosis, lipid uptake and metabolism, phagocytosis, long term potentiation, cell migration, telomere maintenance, and angiogenesis. Moreover, water-soluble derivatives of vitamin E including some based on αTP are increasingly used as components of nanocarriers for enhanced and targeted delivery of drugs and other molecules (vitamins, including αT and αTP itself, vitamin D3, carnosine, caffeine, docosahexaenoic acid (DHA), insulin) and cofactors such as coenzyme Q10. In this review, the chemical characteristics, transport, metabolic pathways, and molecular mechanisms of action of αTP in cells and tissues are summarized and put into perspective with its possible role in the prevention of a number of diseases.

α-Tocopheryl Phosphate Induces VEGF Expression via CD36/PI3Kγ in THP-1 Monocytes.

The CD36 scavenger receptor binds several ligands and mediates ligand uptake and ligand-dependent signal transduction and gene expression, events that may involve CD36 internalization. Here we show that CD36 internalization in THP-1 monocytes is triggered by α-tocopherol (αT) and more strongly by α-tocopheryl phosphate (αTP) and EPC-K1, a phosphate diester of αTP and L-ascorbic acid. αTP-triggered CD36 internalization is prevented by the specific covalent inhibitor of selective lipid transport by CD36, sulfo-N-succinimidyl oleate (SSO). Moreover, SSO inhibited the CD36-mediated uptake of 14C-labelled αTP suggesting that αTP binding and internalization of CD36 is involved in cellular αTP uptake, whereas the uptake of αT was less affected. Similar to that, inhibition of selective lipid transport of the SR-BI scavenger receptor resulted mainly in reduction of αTP and not αT uptake. In contrast, uptake of αT was mainly inhibited by Dynasore, an inhibitor of clathrin-mediated endocytosis, suggesting that the differential regulatory effects of αTP and αT on signaling may be influenced by their different routes of uptake. Interestingly, αTP and EPC-K1 also reduced the neutral lipid content of THP-1 cells and the phagocytosis of fluorescent Staphylococcus aureus bioparticles. Moreover, induction of the vascular endothelial growth factor (VEGF) promoter activity by αTP occurred via CD36/PI3Kγ/Akt, as it could be inhibited by specific inhibitors of this pathway (SSO, Wortmannin, AS-605240). These results suggest that αTP activates PI3Kγ/Akt signaling leading to VEGF expression in monocytes after binding to and/or transport by CD36, a receptor known to modulate angiogenesis in response to amyloid beta, oxLDL, and thrombospondin. J. Cell. Biochem. 118: 1855-1867, 2017. © 2017 Wiley Periodicals, Inc.

Modulation of cAMP levels by high-fat diet and curcumin and regulatory effects on CD36/FAT scavenger receptor/fatty acids transporter gene expression.

Curcumin, a polyphenol from turmeric (Curcuma longa), reduces inflammation, atherosclerosis, and obesity in several animal studies. In Ldlr-/- mice fed a high-fat diet (HFD), curcumin reduces plasma lipid levels, therefore contributing to a lower accumulation of lipids and to reduced expression of fatty acid transport proteins (CD36/FAT, FABP4/aP2) in peritoneal macrophages. In this study, we analyzed the molecular mechanisms by which curcumin (500, 1000, 1500 mg/kg diet, for 4 months) may influence plasma and tissue lipid levels in Ldlr-/- mice fed an HFD. In liver, HFD significantly suppressed cAMP levels, and curcumin restored almost normal levels. Similar trends were observed in adipose tissues, but not in brain, skeletal muscle, spleen, and kidney. Treatment with curcumin increased phosphorylation of CREB in liver, what may play a role in regulatory effects of curcumin in lipid homeostasis. In cell lines, curcumin increased the level of cAMP, activated the transcription factor CREB and the human CD36 promoter via a sequence containing a consensus CREB response element. Regulatory effects of HFD and Cur on gene expression were observed in liver, less in skeletal muscle and not in brain. Since the cAMP/protein kinase A (PKA)/CREB pathway plays an important role in lipid homeostasis, energy expenditure, and thermogenesis by increasing lipolysis and fatty acid ß-oxidation, an increase in cAMP levels induced by curcumin may contribute to its hypolipidemic and anti-atherosclerotic effects. © 2016 BioFactors, 43(1):42-53, 2017.

Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.

The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.