In vitro evaluation of chitosan copper chelate gels as a multimicronutrient feed additive for cattle.

BACKGROUND: Effective micronutrient supplementation strategies are critical to ensure optimal health and productivity in livestock. The objective of this study was to develop a copper and vitamin (multimicronutrient) delivery system based on chitosan gel beads, and test its suitability, in vitro, for use as a cattle feed additive. RESULTS: Chitosan was chelated with copper sulfate to produce millimetre-scale gel matrices (∼2 mm). The copper content was significantly increased (from 61 to 95 mg g by adjusting pH to alkaline conditions post bead formation. The beads could subsequently be loaded with the model vitamin riboflavin to levels as high as 324 µg g-1 beads. Restricted rehydration of the dried gel matrices in simulated rumen fluid led to a sustained release of riboflavin with no copper released in these neutral conditions for up to 24 h, demonstrating copper rumen bypass. Moreover, sustained release of the mineral was observed in abomasal conditions of pH 2 over a 3 h period. CONCLUSIONS: The matrices showed rumen bypass for copper yet supplied nutritionally relevant levels of the free mineral in abomasal conditions, as required for effective supplementation in cattle. The controlled-release properties demonstrated by the matrices indicate their potential as a multimicronutrient functional feed additive to enhance cattle nutrition and productivity. © 2018 Society of Chemical Industry.

Copper-Chelated Chitosan Microgels for the Selective Enrichment of Small Cationic Peptides.

Copper-chelated chitosan microgels were investigated as an immobilized metal affinity chromatography (IMAC) phase for peptide separation. The copper-crosslinked chitosan beads were shown to strongly interact with a range of amino acids, in a wide range of pH and saline conditions. The beads exhibited an affinity that seemed to depend on the isoelectric point of the amino acid, with the extent of uptake increasing with decreasing isoelectric point. This selective interaction with anionic amino acids resulted in a significant relative enrichment of the supernatant solution in cationic amino acids. The beads were then studied as a novel fractionation system for complex milk hydrolysates. The copper chitosan beads selectively removed larger peptides from the hydrolysate aqueous solution, yielding a solution relatively enriched in medium and smaller peptides, which was characterized both quantitatively and qualitatively by size exclusion chromatography (SEC). Liquid chromatography-mass spectrometry (LCMS) work provided comprehensive data on a peptide sequence level and showed that a depletion of the anionic peptides by the beads resulted in a relative enrichment of the cationic peptides in the supernatant solution. It could be concluded that after fractionation a dramatic relative enrichment in respect to small- and medium-sized cationic peptides in the solution, characteristics that have been linked to bioactivities, such as anti-microbial and cell-penetrating properties. The results demonstrate the use of the chitosan copper gel bead system in lab scale fractionation of complex hydrolysate mixtures, with the potential to enhance milk hydrolysate bioactivity.

The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer.

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.

Precision medicine by designer interference peptides: applications in oncology and molecular therapeutics.

In molecular cancer therapeutics only 10% of known cancer gene products are targetable with current pharmacological agents. Major oncogenic drivers, such as MYC and KRAS proteins are frequently highly overexpressed or mutated in multiple human malignancies. However, despite their key role in oncogenesis, these proteins are hard to target with traditional small molecule drugs due to their large, featureless protein interfaces and lack of deep pockets. In addition, they are inaccessible to large biologicals, which are unable to cross cell membranes. Designer interference peptides (iPeps) represent emerging pharmacological agents created to block selective interactions between protein partners that are difficult to target with conventional small molecule chemicals or with large biologicals. iPeps have demonstrated successful inhibition of multiple oncogenic drivers with some now entering clinical settings. However, the clinical translation of iPeps has been hampered by certain intrinsic limitations including intracellular localization, targeting tissue specificity and pharmacological potency. Herein, we outline recent advances for the selective inhibition of major cancer oncoproteins via iPep approaches and discuss the development of multimodal peptides to overcome limitations of the first generations of iPeps. Since many protein-protein interfaces are cell-type specific, this approach opens the door to novel programmable, precision medicine tools in cancer research and treatment for selective manipulation and reprogramming of the cancer cell oncoproteome.

Honeybee venom and melittin suppress growth factor receptor activation in HER2-enriched and triple-negative breast cancer.

Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee (Apis mellifera) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the anticancer selectivity of melittin, and outlines treatment strategies to target aggressive breast cancers.

Investigating normal day to day variations of postural control in a healthy young population using Wii balance boards.

The quantification of postural control (PC) provides the opportunity to understand the function and integration of the sensorimotor subsystems. The increased availability of portable sensing technology, such as Wii Balance Boards (WBB), has afforded the capacity to capture data pertaining to motor function, outside of the laboratory and clinical setting. However, prior to its use in long-term monitoring, it is crucial to understand natural daily PC variation. Twenty-four young adults conducted repeated static PC assessments over 20 consecutive weekdays, using WBBs. 16/24 participants (eyes open) and 11/24 participants (eyes closed) exhibited statistically significant differences (p <; 0.05) between their initial `once-off' measure and their daily measures of PC. This study showed that variations in PC exist in a healthy population, a once-off measure may not be representative of true performance and this inherent variation should be considered when implementing long-term monitoring protocols.

Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system.

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.