Albumin-Binding Lutetium-177-Labeled LLP2A Derivatives as Theranostics for Melanoma.

Very late antigen-4 (VLA-4) is a transmembrane integrin protein that is highly expressed in aggressive forms of metastatic melanoma. A small-molecule peptidomimetic, LLP2A, was found to have a low pM affinity binding to VLA-4. Because LLP2A itself does not inhibit cancer cell proliferation and survival, it is an ideal candidate for the imaging and delivery of therapeutic payloads. An analog of [177Lu]Lu-labeled-LLP2A was previously investigated as a therapeutic agent in melanoma tumor-bearing mice, resulting in only a modest improvement in tumor growth inhibition, likely due to rapid clearance of the agent from the tumor. To improve the pharmacokinetic profile, DOTAGA-PEG4-LLP2A with a 4-(p-iodophenyl)butyric acid (pIBA) albumin binding moiety was synthesized. We demonstrate the feasibility of this albumin binding strategy by comparing in vitro cell binding assays and in vivo biodistribution performance of [177Lu]Lu-DOTAGA-PEG4-LLP2A ([177Lu]Lu-1) to the albumin binding [177Lu]Lu-DOTAGA-pIBA-PEG4-LLP2A ([177Lu]Lu-2). In vitro cell binding assay results for [177Lu]Lu-1 and [177Lu]Lu-2 showed Kd values of 0.40 ± 0.07 and 1.75 ± 0.40 nM, with similar Bmax values of 200 ± 6 and 315 ± 15 fmol/mg, respectively. In vivo biodistribution data for both tracers exhibited specific uptake in the tumor, spleen, thymus, and bone due to endogenous expression of VLA-4. Compound [177Lu]Lu-2 exhibited a much longer blood circulation time compared to [177Lu]Lu-1. The tumor uptake for [177Lu]Lu-1 was highest at 1 h (∼15%ID/g) and that for [177Lu]Lu-2 was highest at 4 h (∼23%ID/g). Significant clearance of [177Lu]Lu-1 from the tumor occurs at 24 h (<5%ID/g) while[177Lu]Lu-2 is retained for greater than 96 h (∼10%ID/g). An efficacy study showed that melanoma tumor-bearing mice receiving compound [177Lu]Lu-2 given in two fractions (2 × 14.8 MBq, 14 days apart) had a greater median survival time than mice administered a single 29.6 MBq dose of compound [177Lu]Lu-1, while a single 29.6 MBq dose of [177Lu]Lu-2 imparted hematopoietic toxicity. The in vitro and in vivo data show addition of pIBA to [177Lu]Lu-DOTAGA-PEG4-LLP2A slows blood clearance for a higher tumor uptake, and there is potential of [177Lu]Lu-2 as a theranostic in fractionated administered doses.

Quantification and characterization of biological activities of glansreginin A in black walnuts (Juglans nigra).

Glansreginin A has been reported to be an indicator of the quality of walnuts (Juglans spp.). However, bioactive properties of glansreginin A have not been adequately explored. In the present study, we quantified concentrations of glansreginin A in black walnuts (Juglans nigra) using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed an array of in vitro bioassays to characterize biological activities (e.g., antibacterial, antioxidant, anticancer capacities) of this compound. Results from HPLC-MS/MS analysis indicated that glansreginin A was presented in all 12 black cultivars examined and its contents were variable among black walnut cultivars, ranged from 6.8 mg/kg (Jackson) to 47.0 mg/kg (Hay). Glansreginin A possessed moderate antibacterial activities against Gram-positive pathogens (Staphylococcus aureus and Bacillus anthracis). This compound exhibited no antioxidant activities, did not induce the activity of antioxidant response element signaling pathways, and exerted no antiproliferative effects on tumorigenic alveolar epithelial cells and non-tumorigenic lung fibroblast cells.

Quantification of Vitamins, Minerals, and Amino Acids in Black Walnut (Juglans nigra).

This paper aims to quantify the micronutrients in black walnut and address its human health benefits. The metabolic profiling of 11 black walnut cultivars was accomplished using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight high-resolution mass spectrometer. Results revealed that the highest concentration of vitamin B9 was present in cultivar "Daniel" (avg. relative signal intensity 229.53 × 104 mAU). "Surprise" and "Daniel" cultivars had the highest amount of vitamin B5. However, vitamin A, D3, E, and K showed no significant difference among the cultivars. The vitamin content levels among the cultivars were compared by applying one way ANOVA method with (P < 0.05) significance level. Mineral analysis for the black walnut kernel, Persian walnut, and black walnut protein powder was done using Inductively Coupled Plasma Optical Emission spectroscopy. The experimental data for black walnut kernel is 0.04 mg/g for Fe and 0.03 mg/g for Zn, and for black walnut, protein powder is 0.07 mg/g for Fe and 0.07 mg/g for Zn. The amino acid analysis and comparison with black walnut kernel show that black walnut flour and protein powder have a higher amount of essential and non-essential amino acids. Therefore, researchers, food process engineers, and food product developers should consider the health benefits of black walnuts and explore the commercial potential of this native agroforestry crop.

Detection of Atrazine and Its Metabolites in Natural Water Samples Using Photonic Molecularly Imprinted Sensors.

In a previous study, photonic-based molecularly imprinted polymers (MIPs) were fabricated using atrazine (ATZ) and its metabolites, desethylatrazine (DEA) and desisopropylatrazine (DIA), as templates in separate matrices. For the purposes of monitoring the abovementioned molecules in natural waters, the effect of natural waters-featuring ionic strength and natural organic matter (NOM) on atrazine MIP-were studied in this work, and the photonic MIP was implemented for monitoring the target molecules in natural water samples collected from land in nearby farms in northeast of Columbia MO. Non-imprinted polymers (NIP) were also fabricated and applied in the experiments as a control test. In presence of NaCl, CaCl2, and NOM, MIPs presented lower responses by 26%, higher responses by 23%, and higher responses by 35%, respectively. NIPs response in terms of an increase or decrease was consistent with those of MIPs, but only for a lower percentage. MIPs response in natural waters-which were characterized for their physicochemical characteristics such as conductivity, total organic carbon content, etc.-provided a good approximation of the real concentrations obtained from the LCMS instrument; in general, they showed a good concordance, although large discrepancies occurred for some samples, which can be related to reproducibility issues in the manufacturing process or the presence of unknown interfering compounds in the real samples.

Toxicometabolomic profiling of resistant and susceptible western corn rootworm larvae feeding on Bt maize seedlings.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most serious pest of maize (Zea mays L.) in the U.S. Corn Belt and parts of Europe. Transgenic maize hybrids expressing at least one of the four currently available insecticidal toxins from Bacillus thuringiensis (Bt) Berliner, currently the most widely adopted control method in continuous maize, have faltered due to the emergence of resistance. The resistance mechanisms of WCR to Bt toxins are not fully understood. We identified metabolic profiles of susceptible and resistant WCR larvae fed on maize hybrids expressing each of three available Cry3 proteins (eCry3Ab1, mCry3A, and Cry3Bb1) targeting corn rootworms and a control non-Bt maize via an untargeted metabolomics approach. Over 580 unique metabolites found in WCR larvae were classified into different pathways (amino acids, carbohydrates, cofactors and vitamins, energy, lipid, nucleotide, peptide, and xenobiotics). By exploring shifts in WCR larval metabolome exclusively by Bt toxins, several candidate metabolites and metabolic pathways were identified in susceptible and resistant larvae that may be involved in defense against or recovery from Bt ingestion by these larvae. These findings would provide mechanistic insights into altered metabolic pathways associated with the resistance mechanisms of WCR to Bt toxins.

p-NCS-Bn-NODAGA as a bifunctional chelator for radiolabeling with the 186Re/99mTc-tricarbonyl core: Radiochemistry with model complexes and a GRPR-targeting peptide.

INTRODUCTION: With the goal of developing theranostic agents for application in radiopharmaceutical chemistry, in this work, we studied p-NCS-Bn-NODAGA (1) as a bifunctional chelator for the fac-[M(CO)3]+ core (M = natRe, 186Re, 99mTc). Specifically, we studied complexes of the formula [M(CO)3(L)]+, where L denotes either Bn-NODAGA-Pyr (2) or Bn-NODAGA-Ser-Ser-RM2 (3). METHODS: The model bioconjugate molecule 2 was synthesized by conjugating pyrrolidine with 1, while 3 was derived from the conjugation of the gastrin-releasing peptide receptor (GRPR)-targeting peptide Ser-Ser-RM2 with 1. Labeling of 2 and 3 was performed with [M(CO)3(OH2)3]+ (where M = natRe, 186Re, or 99mTc). The stability of the radioactive complexes was studied against l-histidine and l-cysteine (1 mM in PBS; pH 7.4, 37 °C). GRPR affinity of both peptide 3 and its metallated counterpart, Re-3, were determined with in vitro competitive binding assays in GRPR-expressing PC-3 cells using [125I]I-Tyr4-BBN as the competitor. RESULTS: After a thorough radiolabeling optimization process, the [M(CO)3(2)]+ model complexes (M = 186Re and 99mTc) were synthesized with 94 ± 2% radiochemical yield (RCY; estimated by radio-HPLC). In stability studies, [186Re]Re-2 remained intact through 7 d in l-cysteine and l-histidine. Similarly, stability studies in rat serum at 37 °C showed 99 ± 1% intact [186Re]Re-2 through 4 h. Non-specific rat serum protein binding of [186Re]Re-2 was found to be 33 ± 4% at 4 h. The [99mTc]Tc-2 complex was found to be stable in l-histidine and l-cysteine at 37 °C through 24 h. [99mTc]Tc-2 was also stable in rat serum, with 38 ± 3% non-specific protein binding, at 4 h. The [M(CO)3(3)]+ peptide radiometal complex (M = 186Re and 99mTc) syntheses were also optimized, resulting in RCYs of 35% for [186Re]Re-3 and 47% for [99mTc]Tc-3 (estimated by radio-HPLC). [186Re]Re-3 showed 98 ± 2% and 84 ± 5% stability in l-histidine and l-cysteine, respectively, through 48 h. Similarly, stability studies in rat serum at 37 °C showed 85 ± 3% intact [186Re]Re-3 through 4 h, with 29 ± 7% non-specific protein binding in rat serum. [99mTc]Tc-3 was found to be 84 ± 3% and 82 ± 4% stable in l-histidine and l-cysteine at 24 h, respectively. [99mTc]Tc-3 in rat serum at 37 °C showed 88 ± 2% stability through 4 h, with 25 ± 2% non-specific protein binding. Both 3 and Re-3 demonstrated high GRPR affinity, with IC50 values of 3.1 nM and 3.9 nM, respectively. CONCLUSIONS: The low nanomolar IC50 values obtained for 3 and Re-3 demonstrate high affinity of this novel [M(CO)3]-labeled bioconjugate for GRPR. The encouraging stability studies and receptor affinity results demonstrate promise for further development of these metal complexes as a theranostic matched pair for targeting GRPR.

PET Imaging of VLA-4 in a New BRAFV600E Mouse Model of Melanoma.

PURPOSE: Despite unprecedented responses to immune checkpoint inhibitors and targeted therapy in melanoma, a major subset of patients progresses and have few effective salvage options. We have previously demonstrated robust, selective uptake of the peptidomimetic LLP2A labeled with Cu-64 ([64Cu]-LLP2A) for positron emission tomography (PET) imaging in subcutaneous and metastatic models of B16F10 murine melanoma. LLP2A binds with high affinity to very late antigen-4 (VLA-4, integrin α4ß1), a transmembrane protein overexpressed in melanoma and other cancers that facilitates tumor growth and metastasis. Yet B16F10 fails to faithfully reflect human melanoma biology, as it lacks certain oncogenic driver mutations, including BRAF mutations found in ≥ 50 % of clinical specimens. Here, we evaluated the PET tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in novel, translational BRAFV600E mutant melanoma models differing in VLA-4 expression-BPR (VLA-4-) and BPRα (VLA-4+). PROCEDURES: BPR cells were transduced with α4 (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [64Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [64Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells was quantified via a plate-based assay. Tracer biodistribution and PET/CT imaging were evaluated in mice bearing subcutaneous BPR and BPRα tumors. RESULTS: [64Cu]-LLP2A demonstrated high binding affinity to BPRα (Kd = 1.4 nM) but indeterminate binding to BPR cells. VLA-4+ BPRα and B16F10 displayed comparable time-dependent [64Cu]-LLP2A internalization, whereas BPR internalization was undetectable. PET/CT showed increased tracer uptake in BPRα tumors vs. BPR tumors in vivo, which was validated by significantly greater (p < 0.0001) BPRα tumor uptake in biodistribution analyses. CONCLUSIONS: [64Cu]-LLP2A discriminates BPRα (VLA-4+) vs. BPR (VLA-4-) melanomas in vivo, supporting translation of these BRAF-mutated melanoma models via prospective imaging and theranostic studies. These results extend the utility of LLP2A to selectively target clinically relevant and therapy-resistant tumor variants toward its use for therapeutic patient care.

Detection of chlorantraniliprole residues in tomato using field-deployable MIP photonic sensors.

A photonic sensor based on inversed opal molecular imprinted polymer (MIP) film to detect the presence of chlorantraniliprole (CHL) residue in tomatoes was developed. Acrylic acid was polymerized in the presence of CHL inside the structure of a colloidal crystal, followed by etching of the colloids and CHL elution. Colloidal crystals and MIP films were characterized by scanning electron microscopy and FT-IR, confirming the inner structure and chemical structure of the material. MIP films supported on polymethylmethacrylate (PMMA) slides were incubated in aqueous solutions of the pesticide and in blended tomato samples. The MIP sensor displayed shifts of the peak wavelength of the reflection spectra in the visible range when incubated in CHL concentrations between 0.5 and 10 µg L-1, while almost no peak displacement was observed for non-imprinted (NIP) films. Whole tomatoes were blended into a liquid and spiked with CHL; the sensor was able to detect CHL residues down to 0.5 µg kg-1, significantly below the tolerance level established by the US Environmental Protection Agency of 1.4 mg kg-1. Stable values were reached after about 30-min incubation in test samples. Control samples (unspiked processed tomatoes) produced peak shifts both in MIP and NIP films; however, this matrix effect did not affect the detection of CHL in the spiked samples. These promising results support the application of photonic MIP sensors as an economical and field-deployable screening tool for the detection of CHL in crops.

Profiling Anticancer and Antioxidant Activities of Phenolic Compounds Present in Black Walnuts (Juglans nigra) Using a High-Throughput Screening Approach.

Our recent studies have demonstrated multiple health-promoting benefits from black walnut kernels. These biological functions of black walnuts are likely associated with their bioactive constituents. Characterization of phenolic compounds found in black walnut could point out underexplored bioactive activities of black walnut extracts and promote the development of novel applications of black walnut and its by-products. In the present study, we assessed bioactivity profiles of phenolic compounds identified in the kernels of black walnuts using a high-throughput screening (HTS) approach. Black walnut phenolic compounds were evaluated in terms of their total antioxidant capacity, antioxidant response element (ARE) induction, and anticancer activities. The anticancer activities were identified by evaluating the effects of the phenolic compounds on the growth of the tumorigenic alveolar epithelial cells (A549) and non-tumorigenic lung fibroblast cells (MRC-5). Out of 16 phenolic compounds tested, several compounds (penta-O-galloyl-ß-d-glucose, epicatechin gallate, quercetin, (-)-epicatechin, rutin, quercetin 3-ß-d-glucoside, gallic acid, (+)-catechin, ferulic acid, syringic acid) exerted antioxidant activities that were significantly higher compared to Trolox, which was used as a control. Two phenolic compounds, penta-O-galloyl-ß-d-glucose and quercetin 3-ß-d-glucoside, exhibited antiproliferative activities against both the tumorigenic alveolar epithelial cells (A549) and non-tumorigenic lung fibroblast cells (MRC-5). The antioxidant activity of black walnut is likely driven not only by penta-O-galloyl-ß-d-glucose but also by a combination of multiple phenolic compounds. Our findings suggested that black walnut extracts possibly possess anticancer activities and supported that penta-O-galloyl-ß-d-glucose could be a potential bioactive agent for the cosmetic and pharmaceutical industries.

Identification and Quantification of Bioactive Molecules Inhibiting Pro-inflammatory Cytokine Production in Spent Coffee Grounds Using Metabolomics Analyses.

In this study, we assessed the anti-inflammatory properties of spent coffee grounds. Methanolic extracts of spent coffee grounds obtained from 3 Arabica cultivars possess compounds that exerted inhibitory effects on the secretion of inflammatory mediators (TNF-α, IL-6, and IL-10) induced by a human pro-monocytic cell line differentiated with PMA and stimulated with lipopolysaccharide (LPS). Our results indicated that the cytokine suppressive activities of the spent coffee ground (SCG) extracts were different among coffee cultivars tested. Hawaiian Kona extracts exhibited inhibitory effects on the expression of 3 examined cytokines, Ethiopian Yirgacheffe extracts reduced the secretion of TNF-α and IL-6, and Costa Rican Tarrazu extracts decreased the secretion of IL-6 only. Untargeted metabolomics analyses of SCG extracts led to the putative identification of 26 metabolites with known anti-inflammatory activities. Multiple metabolites (i.e., chrysin, daidzein, eugenol, naringenin, naringin, oxyresveratrol, pectolinarin, resveratrol, tectochrysin, theaflavin, vanillic acid, and vitexin rhamnoside) identified in the SCGs represent possible novel anti-inflammatory compounds. Of the 26 identified metabolites, the 12 compounds that had high relative intensities in all of the extracts were successfully quantified using liquid chromatography-tandem mass spectrometry analyses. Results from the targeted analyses indicated that caffeine and 5-caffeoylquinic acid (CQA) were the most abundant compounds in the SCG extracts. The contents of caffeine ranged from 0.38 mg/g (Ethiopian Yirgacheffe) - 0.44 mg/g (Costa Rican Tarrazu), whereas 5-CQA concentrations were in the range of 0.24 mg/g (Costa Rican Tarrazu) - 0.34 mg/g (Ethiopian Yirgacheffe). The presence of multiple anti-inflammatory compounds in SCGs provides a promising natural source for cosmetic and pharmaceutical industries.

Black Walnut (Juglans nigra) Extracts Inhibit Proinflammatory Cytokine Production From Lipopolysaccharide-Stimulated Human Promonocytic Cell Line U-937.

Black walnut (Juglans nigra L.) is an excellent source of health-promoting compounds. Consumption of black walnuts has been linked to many health benefits (e.g., anti-inflammatory) stemming from its phytochemical composition and medicinal properties, but these effects have not been systematically studied or characterized. In this study, potential anti-inflammatory compounds found in kernel extracts of 10 black walnut cultivars were putatively identified using a metabolomic profiling analysis, revealing differences in potential anti-inflammatory capacities among examined cultivars. Five cultivars were examined for activities in the human promonocytic cell line U-937 by evaluating the effects of the extracts on the expression of six human inflammatory cytokines/chemokines using a bead-based, flow cytometric multiplex assay. The methanolic extracts of these cultivars were added at four concentrations (0.1, 0.3, 1, and 10 mg/ml) either before and after the addition of lipopolysaccharide (LPS) to human U-937 cells to examine their effect on cytokine production. Results from cytotoxicity and viability assays revealed that the kernel extracts had no toxic effect on the U-937 cells. Of the 13 cytokines [interleukin (IL)-1ß, tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein (MCP)-1, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IL-23, IL-33, interferon (IFN)-α, IFN-γ] measured, only six were detected under the culture conditions. The production of the six detected cytokines by phorbol 12-myristate 13-acetate (PMA)-differentiated, LPS-stimulated U-937 was significantly inhibited by the kernel extracts from two cultivars Surprise and Sparrow when the extracts were added before the addition of LPS. Other cultivars (Daniel, Mystry, and Sparks) showed weak or no significant effects on cytokine production. In contrast, no inhibitory effect was observed on the production of cytokines by PMA-differentiated, LPS-stimulated U-937 when the kernel extracts were added after the addition of LPS. The findings suggest that the extracts from certain black walnut cultivars, such as Sparrow and Surprise, are promising biological candidates for potentially decreasing the severity of inflammatory disease.

Identifying Antibacterial Compounds in Black Walnuts (Juglans nigra) Using a Metabolomics Approach.

Black walnut (Juglans nigra L.) is one of the most economically valuable hardwood species and a high value tree for edible nut production in the United States. Although consumption of black walnut has been linked to multiple health-promoting effects (e.g., antioxidant, antimicrobial, anti-inflammatory), the bioactive compounds have not been systematically characterized. In addition, the associations between different black walnut cultivars and their health-promoting compounds have not been well established. In this study, the kernels of twenty-two black walnut cultivars selected for nut production by the University of Missouri Center for Agroforestry (Columbia, MO, USA) were evaluated for their antibacterial activities using agar-well diffusion assay. Among the selected cultivars, four black walnut cultivars (i.e., Mystry, Surprise, D.34, and A.36) exhibited antibacterial activity against a Gram-positive bacterium (Staphylococcus aureus), whereas other cultivars showed no effect on the inhibition of this bacterium. The antibacterial compounds showing the strongest activity were isolated with bioassay-guided purification and identified using a metabolomics approach. Six antibacterial bioactive compounds responsible for antimicrobial activity were successfully identified. Glansreginin A, azelaic acid, quercetin, and eriodictyol-7-O-glucoside are novel antibacterial compounds identified in the kernels of black walnuts. The metabolomics approach provides a simple and cost-effective tool for bioactive compound identification.