Genetic dissection of grain iron and zinc, and thousand kernel weight in wheat (Triticum aestivum L.) using genome-wide association study.

Genetic biofortification is recognized as a cost-effective and sustainable strategy to reduce micronutrient malnutrition. Genomic regions governing grain iron concentration (GFeC), grain zinc concentration (GZnC), and thousand kernel weight (TKW) were investigated in a set of 280 diverse bread wheat genotypes. The genome-wide association (GWAS) panel was genotyped using 35 K Axiom Array and phenotyped in five environments. The GWAS analysis showed a total of 17 Bonferroni-corrected marker-trait associations (MTAs) in nine chromosomes representing all the three wheat subgenomes. The TKW showed the highest MTAs (7), followed by GZnC (5) and GFeC (5). Furthermore, 14 MTAs were identified with more than 10% phenotypic variation. One stable MTA i.e. AX-95025823 was identified for TKW in both E4 and E5 environments along with pooled data, which is located at 68.9 Mb on 6A chromosome. In silico analysis revealed that the SNPs were located on important putative candidate genes such as Multi antimicrobial extrusion protein, F-box domain, Late embryogenesis abundant protein, LEA-18, Leucine-rich repeat domain superfamily, and C3H4 type zinc finger protein, involved in iron translocation, iron and zinc homeostasis, and grain size modifications. The identified novel MTAs will be validated to estimate their effects in different genetic backgrounds for subsequent use in marker-assisted selection. The identified SNPs will be valuable in the rapid development of biofortified wheat varieties to ameliorate the malnutrition problems.

Facile Bottom-up Preparation of WS2-Based Water-Soluble Quantum Dots as Luminescent Probes for Hydrogen Peroxide and Glucose.

Photoluminescent zero-dimensional (0D) quantum dots (QDs) derived from transition metal dichalcogenides, particularly molybdenum disulfide, are presently in the spotlight for their advantageous characteristics for optoelectronics, imaging, and sensors. Nevertheless, up to now, little work has been done to synthesize and explore photoluminescent 0D WS2 QDs, especially by a bottom-up strategy without using usual toxic organic solvents. In this work, we report a facile bottom-up strategy to synthesize high-quality water-soluble tungsten disulfide (WS2) QDs through hydrothermal reaction by using sodium tungstate dihydrate and L-cysteine as W and S sources. Besides, hybrid carbon quantum dots/WS2 QDs were further prepared based on this method. Physicochemical and structural analysis of QD hybrid indicated that the graphitic carbon quantum dots with diameters about 5 nm were held onto WS2 QDs via electrostatic attraction forces. The resultant QDs show good water solubility and stable photoluminescence (PL). The excitation-dependent PL can be attributed to the polydispersity of the synthesized QDs. We found that the PL was stable under continuous irradiation of UV light but can be quenched in the presence of hydrogen peroxide (H2O2). The obtained WS2-based QDs were thus adopted as an electrodeless luminescent probe for H2O2 and for enzymatic sensing of glucose. The hybrid QDs were shown to have a more sensitive LOD in the case of glucose sensing. The Raman study implied that H2O2 causes the partial oxidation of QDs, which may lead to oxidation-induced quenching. Overall, the presented strategy provides a general guideline for facile and low-cost synthesis of other water-soluble layered material QDs and relevant hybrids in large quantity. These WS2-based high-quality water-soluble QDs should be promising for a wide range of applications in optoelectronics, environmental monitoring, medical imaging, and photocatalysis.

Comparative Photothermal Performance among Various Sub-Stoichiometric 2D Oxygen-Deficient Molybdenum Oxide Nanoflakes and In†Vivo Toxicity.

The present study deals with photothermal therapy of solid tumors using different forms of oxygen-deficient sub-stoichiometric two-dimensional (2D) molybdenum oxide nanoflakes (α-MoO3-x ). Upon exfoliation of molybdenum oxide power using fine gridding followed by ultrasonication, bluish green molybdenum oxide (BG α-MoO3 ) was obtained. Oxygen vacancies in BG were generated upon irradiation with an intense xenon lamp. Irradiating the BG for 3 and 5 h, deep blue (B) and olive green (G) oxygen-deficient nanoflakes were obtained respectively. All exhibited high NIR absorption, making these nanomaterials suitable for photothermal therapy. All three forms were functionalized with polypyrrole (PPy@BG, PPy@B, PPy@G) to boost the photothermal stability and transduction efficiency. After functionalization and irradiation with 808 nm laser, the enhancement of temperature for BG, B, G was 50, 65, 52 °C respectively and the corresponding photothermal transduction efficiencies (PTE) were 29.32, 44.42 and 42.00 %. Each of the nanoflakes were found to be highly biocompatible and photostable both in vitro and in vivo. There was substantial decrease in the size of tumors after seven days of treatment on tumor-bearing experimental mice models.

Expression, characterization, and evaluation of a RANK-binding single chain fraction variable: an osteoclast targeting drug delivery strategy.

A single chain Fraction variable (scFv) employs antibody-like target recognition specificity. Osteoclasts, responsible for bone resorption, express Receptor Activator of Nuclear factor Kappa B (RANK) receptors. This study aimed to express, characterize, and evaluate scFv against RANK receptors that may serve as a platform to target osteoclasts. Using phage display technology, scFv against RANK receptor was expressed and characterized by DNA sequencing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption-ionization time-of-flight (MALDI TOF), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunocytochemistry. The potential for cytotoxicity was evaluated using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, and its cross reactivity was evaluated using ELISA. Osteoclast-like cells were generated from RAW 264.7 cells, and the osteoclast targeting ability of scFv was evaluated using immunocytochemistry. ScFv's antiresorptive efficacy was studied using a tartrate-resistant acid phosphatase (TRAP) assay and resorption assay. Anti-RANK scFv was successfully expressed and characterized. No cross reactivity with other tumor necrosis factor receptor (TNFR) members and no cytotoxic effect on a non-RANK bearing cell line were observed. It showed specificity toward a RANK receptor and an inhibitory effect on osteoclast activity. With the increase in development trends for biologics as therapeutics and growing knowledge on the importance of osteoclast targeted therapy, this study may provide a drug delivery strategy to target osteoclasts, thereby leading to a promising therapy for resorptive bone diseases.

Synthesis, characterization and evaluation of bone targeting salmon calcitonin analogs in normal and osteoporotic rats.

In order to assess the therapeutic efficacy of an antiresorptive drug with imparted bone targeting potential using bisphosphonate (BP) conjugation and an improved pharmacokinetic profile using PEGylation, we synthesized, characterized and evaluated in vivo efficacy of bone-targeting PEGylated salmon calcitonin (sCT) analog (sCT-PEG-BP). sCT-PEG-BP was compared with non-PEGylated bone targeting sCT analog (sCT-BP) and unmodified, commercially available sCT. sCT-PEG-BP conjugates were characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis. The effect of PEG-BP or BP upon sCT secondary structure was examined by Circular Dichroism and sCT-PEG-BP was evaluated for in vitro bone mineral Hydroxyapatite (HA) binding ability and calcium salts specificity using a binding assay for bone HA and several calcium salts. Anti-calcitonin antibody binding ability of these analogs was determined using enzyme-linked immunosorbent assay (ELISA), by reacting bone targeting sCT analogs with calcium phosphate coated Osteologic® plates and detecting the bound sCT using anti-sCT antibody. Potential cytotoxicity of these compounds was evaluated in monocytic RAW 264.7 cells, and sCT bioactivity was evaluated using an in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells. Finally, in vivo efficacy of each compound was evaluated by determining the plasma levels of calcium after s.c. administration in normal rats, and in a rat model of Osteoporosis, secondary to ovariectomy (OVX). In vivo micro-computed tomography (micro-CT) was used to temporally map and quantify alterations in bone volume and bone mineral density (BMD) in the same animals at 1, 4, 8 and 12 weeks after OVX surgery. Sixteen 6 week old virgin female rats underwent OVX surgery followed by the daily s.c. injection of 2.5IU/kg/day sCT or equivalent analogs, and compared to four sham-operated, placebo treated control rats. Our results showed the chemical coupling of PEG-BP or BP to sCT altered its secondary structure without altering its antibody binding ability. sCT analogs retained strong sCT bioactivity, were non-toxic to RAW 264.7 cells in culture and elicited a comparable hypocalcemic effect to that of unmodified sCT in normal rats. Compared to marketed unmodified sCT, sCT-PEG-BP showed significantly improved efficacy in terms of preserving bone volume, BMD and trabecular micro-architecture in osteoporotic rats at the initial dose tested. Bisphosphonate-mediated targeting of PEGylated sCT to bone represents a new class of targeted antiresorptive compounds that has not previously been attempted.

Antibody-mediated "universal" osteoclast targeting platform using calcitonin as a model drug.

PURPOSE: To generate and characterize a specific monoclonal antibody (mAb) against recombinant human RANK receptor and to develop an antiresorptive strategy using this mAb as an osteoclast-targeting platform that selectively targets osteoclast cells whilst delivering an attached (i.e. chemically conjugated) active drug cargo. METHODS: Using hybridoma technology, we generated a specific monoclonal antibody (mAb) against recombinant human RANK receptor and characterized by SDS PAGE, ELISA, Western Blot and immunocytochemistry, then synthesized osteoclast-targeting bioconjugates of salmon calcitonin (sCT) using this antibody by generating thiol groups on mAb using 2-Iminothiolane and subsequently reacting them with sCT-PEG-MAL synthesised from sCT and NHS-PEG-MAL. To test the efficacy of the conjugate in vitro, osteoclasts were generated from precursor RAW 264.7 cells by dosing with the cytokines macrophage-colony-stimulating factor (M-CSF), and RANK Ligand (RANKL) and TRAP activity assay, Resorption Pit Assay, TRAP staining were performed. Cytotoxicity of the mAb-sCT conjugate was also evaluated in RAW 264.7 cells; sCT bioactivity and CTR binding potential were evaluated by in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells. RESULTS: Generation of antibody against human RANK receptor was confirmed by SDS PAGE, ELISA and Western Blot. Immunocytochemistry confirmed the osteoclast targeting potential of the antibody. Successful conjugation of the antibody with sCT was confirmed by SDS PAGE and ELISA.Multinucleated osteoclast formation was confirmed by staining for tartrate-resistant acid phosphatase (TRAP). Conjugate functionality was confirmed by TRAP activity and Resorption Pit assay, showing the inhibitory effect on osteoclast differentiation. cAMP assay confirmed the retention of calcitonin bioactivity after conjugation. CONCLUSIONS: Our strategy offers the potential for a "universal" osteoclast-targeting platform--one that targets the RANK receptor on osteoclast cells by simply altering the conjugated cargo in order to affect the specific regulation of osteoclast cells.

Synthesis, characterization and in vitro evaluation of a bone targeting delivery system for salmon calcitonin.

Synthetic salmon Calcitonin (sCT) is currently used to treat and manage conditions associated with low bone mass, and elicits its antiresorptive effect by acting upon Calcitonin receptors (CTRs) located on bone-resorbing osteoclast cells. However, CTRs are also widely distributed in many non-skeletal tissues (such as kidney, brain, and lung), and the competitive uptake of available sCT amongst such CTRs likely reduces sCT availability for bone resident osteoclast cells, particularly if the drug is administered systemically and not specifically targeted to bone. Hence, the objective of this study was to synthesize and characterize a bisphosphonate (BP)-mediated bone targeting delivery system for sCT and to determine whether the bioactivity of sCT was retained after BP conjugation. BP-sCT conjugates were synthesized by initially reacting sCT with sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) in dimethyl formamide in the presence of triethylamine (TEA) at room temperature. Thiolated (Thiol)-BP was then reacted with the sCT-sulfo-SMCC conjugates to generate sCT-BP conjugates, which were purified by dialysis and assayed using the micro-BCA protein assay. Non-BP containing control sCT-Cysteine conjugates were also synthesized using the same procedure. Reactions were monitored and characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF) analysis and Tris-Tricine SDS-PAGE. Conjugates were evaluated for in vitro bone mineral affinity using a hydroxyapatite binding test, for bone mineral specificity using different calcium salt binding affinity assays, and for continued sCT bioactivity after conjugation using an intracellular cAMP stimulation in human T47D breast cancer cells. Our results confirmed that BP-conjugated sCT exhibited significantly greater affinity and specificity for bone mineral over unmodified sCT, and that sCT-BP conjugates retained strong CT bioactivity after conjugation. Our conjugation strategy holds the promise of facilitating the delivery of sCT preferentially to skeletal bony tissues, thereby increasing its local concentration to bone surfaces. This peptide hormone-bisphosphonate drug system represents a new class of antiresorptive drug that has not previously been attempted, nor has a bone targeting formulation of sCT been reported.