INVESTIGATION OF THE NONLINEAR INDEX OF REFRACTION OF WATER AT 815 AND 407 NANOMETERS

Using a highly stable spectral interferometry technique, ultrafast processes can be measured within a 2 ps temporal window. The technique was used to measure the nonlinear index of refraction due to the optical Kerr effect in water at both 815 nm and 407 nm with pump pulse lengths of ~90 femtoseconds and ~250 femtoseconds respectively. The 815 nm measurement serves as a benchmark against previous published values while the 407 nm measurement is entirely new. Knowing the value of the nonlinear index at 407 nm allows for pulse tailoring to achieve remote underwater pulse compression and self-focusing

Oscillatory cAMP signaling rapidly alters H3K4 methylation

receptors (GPCRs) alter H3K4 methylation via oscillatory intracellular cAMP. Activation of Gs-coupled receptors caused a rapid decrease of H3K4me3 by elevating cAMP, whereas stimulation of Gi-coupled receptors increased H3K4me3 by diminishing cAMP. H3K4me3 gradually recovered towards baseline levels after the removal of GPCR ligands, indicating that H3K4me3 oscillates in tandem with GPCR activation. cAMP increased intracellular labile Fe(II), the cofactor for histone demethylases, through a non-canonical cAMP target—Rap guanine nucleotide exchange factor-2 (RapGEF2), which subsequently enhanced endosome acidification and Fe(II) release from the endosome via vacuolar H+-ATPase assembly. Removing Fe(III) from the media blocked intracellular Fe(II) elevation after stimulation of Gs-coupled receptors. Iron chelators and inhibition of KDM5 demethylases abolished cAMP-mediated H3K4me3 demethylation. Taken together, these results suggest a novel function of cAMP signaling in modulating histone demethylation through labile Fe(II)

Tissue-Engineered Stromal Reticula to Study Tolerogenic Fibroblastic Reticular Cell Properties in Type I Diabetes

Type 1 Diabetes (T1D) results in β cell destruction from impaired T cell tolerance. Exploiting therapeutic tolerance induction mechanisms using fibroblastic reticular cells (FRC) could protect against the disease. FRCs contribute to peripheral tolerance in lymph nodes (LN) and create supportive, interconnected reticula and allow expansion during inflammation. FRCs also express and present self-antigens, including β cell antigens, to autoreactive T cells. However, unlike professional antigen-presenting cells, FRCs do so with limited co-stimulation, thereby inducing T cell tolerance. In T1D LNs, the FRC frequency, expression of T1D-relevant antigens, and reticular organization decrease, likely reducing FRC tolerogenic engagement of autoreactive T cells. Our goal is to tissue-engineer FRC reticula that recapitulate these organizational changes in inflamed and healthy LNs to determine tolerance mechanisms and develop T1D FRC-based therapies. We previously showed that genetically-engineered FRCs overexpressing T1D-specific autoantigens seeded in collagen scaffolds with 400μm-diameter pores generated reticula with pores recapitulating inflamed LNs (100-200 μm) and engaged antigen-specific T cells. We then fabricated freeze-dried gelatin scaffolds with tunable pore sizes to generate FRC reticula resembling non-inflamed LN pores (15-20µm). The reticular pores of scaffolds frozen in liquid nitrogen had smaller pore sizes (<50µm) compared to -80ºC (<150µm) when characterized by SEM. We confirmed that FRCs seeded in each scaffold form reticula with pore sizes mimicking healthy (37um) and inflamed (89um) LNs via confocal microscopy and remain viable for at least 21 days via CellTiter-Glow assay. We are currently investigating the effects of FRC reticula pore sizes on the engagement of T1D antigen-specific T cells with T1D antigen-expressing FRCs from a T1D mouse model. Our tissue-engineering approach will allow us to study FRC-T cell interactions in an environment that recapitulates the T1D FRC reticula. We ultimately aim to test FRC tolerogenic mechanisms and develop novel FRC-based therapies for peripheral tolerance induction in T1D.</p

Evaluation of fingermark detection sequences on paper substrates

It is generally accepted that the amino acid reagent consisting of 1,2-indanedione and a catalytic amount of zinc chloride, referred to as IND-Zn, is the single best method for the detection of latent fingermarks on paper substrates and that ninhydrin is of limited value when used in sequence after this reagent. However, recent research has suggested that the sequence 1,8-diazafluoren-9-one (DFO) followed by ninhydrin may actually produce a greater number of fingermarks than IND-Zn on its own or IND-Zn followed by ninhydrin.This study focussed on the evaluation of two fingermark detection sequences for porous surfaces: (1) IND-Zn followed by ninhydrin, physical developer (PD) and the lipid stain nile red; and (2) DFO followed by ninhydrin, PD and nile red. The evaluation was undertaken using a range of latent fingermark donors and on a number of paper substrates that are commonly encountered in Australia. In addition, a pseudo-operational trial was completed on 5-year-old university examination booklets. Parallel studies were undertaken at two locations: Sydney (temperate, coastal climate) and Canberra (relatively dry, continental climate).The results of the donor study indicated that there was a negligible difference in performance between the two sequences across all paper types and all time periods evaluated. When considering individual reagents, IND-Zn generally developed better quality fingermarks compared to DFO; however, ninhydrin had a greater enhancement effect on DFO developed marks than after IND-Zn. In the pseudo-operational trials, the IND-Zn sequence outperformed the DFO sequence. Nile red did not develop any additional marks at the end of each sequence and, as a result, the use of this technique at the end of a full sequence is of questionable value.The overall outcome was that the sequence IND-Zn followed by ninhydrin and PD is recommended for the processing of common paper substrates under the conditions typically experienced at the two locations studied. © 2014 Elsevier Ireland Ltd

Ascorbate Suppresses VEGF Expression in Retinal Pigment Epithelial Cells

To investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells. Dot-blot and hydroxymethylated DNA immunoprecipitation sequencing were applied to evaluate the impact of ascorbate on DNA hydroxymethylation in ARPE-19 cells. RNA sequencing (RNA-seq) was carried out to analyze the transcriptome. Quantitative RT-PCR and ELISA were conducted to examine the transcription and secretion of VEGF from cultured cells. Primary human fetal RPE cells and RPE-J cells were used to verify the effect of ascorbate on VEGF expression. ELISA was used to measure VEGF in the vitreous humor of Gulo-/- mice, which, like humans, cannot synthesize ascorbate de novo. Treatment with ascorbate (50 μM) promoted 5-hydroxymethycytosine (5hmC) generation and changed the genome-wide profiles of 5hmC in ARPE-19 cells. Ascorbate also caused a dramatic shift in the transcriptome-3186 differential transcripts, of which 69.3% are correlated with altered 5hmC in promoters or gene bodies. One of the most downregulated genes was VEGFA, which encodes the VEGF protein. The suppression of VEGF by ascorbate is independent of hypoxia-inducible factor 1-alpha (HIF-1α) but correlates with increased 5hmC in the gene body. The decreased transcription and secretion of VEGF by ascorbate were verified in primary human fetal RPE cells. Furthermore, adding ascorbate in the diet for Gulo-/- mice resulted in decreased levels of VEGF in the RPE/choroid and vitreous humor. Ascorbate inhibits VEGF expression in RPE cells likely via DNA hydroxymethylation. Thus, ascorbate could be implicated in the prevention or treatment of diseases such as age-related macular degeneration (AMD)

Vitamin C sensitizes triple negative breast cancer to PI3K inhibition therapy

Rationale: The clinical use of PI3K inhibitors, such as buparlisib, has been plagued with toxicity at effective doses. The aim of this study is to determine if vitamin C, a potent epigenetic regulator, can improve the therapeutic outcome and reduce the dose of buparlisib in treating PIK3CA -mutated triple negative breast cancer (TNBC). Methods: The response of TNBC cells to buparlisib was assessed by EC 50 measurements, apoptosis assay, clonogenic assay, and xenograft assay in mice. Molecular approaches including Western blot, immunofluorescence, RNA sequencing, and gene silencing were utilized as experimental tools. Results: Treatment with buparlisib at lower doses, along with vitamin C, induced apoptosis and inhibited the growth of TNBC cells in vitro . Vitamin C via oral delivery rendered a sub-therapeutic dose of buparlisib able to inhibit TNBC xenograft growth and to markedly block metastasis in mice. We discovered that buparlisib and vitamin C coordinately reduced histone H3K4 methylation by enhancing the nuclear translocation of demethylase, KDM5, and by serving as a cofactor to promote KDM5-mediated H3K4 demethylation. The expression of genes in the PI3K pathway, such as AKT2 and mTOR, was suppressed by vitamin C in a KDM5-dependent manner. Vitamin C and buparlisib cooperatively blocked AKT phosphorylation. Inhibition of KDM5 largely abolished the effect of vitamin C on the response of TNBC cells to buparlisib. Additionally, vitamin C and buparlisib co-treatment changed the expression of genes, including PCNA and FILIP1L, which are critical to cancer growth and metastasis. Conclusion: Vitamin C can be used to reduce the dosage of buparlisib needed to produce a therapeutic effect, which could potentially ease the dose-dependent side effects in patients

Oscillatory cAMP signaling rapidly alters H3K4 methylation.

Epigenetic variation reflects the impact of a dynamic environment on chromatin. However, it remains elusive how environmental factors influence epigenetic events. Here, we show that G protein-coupled receptors (GPCRs) alter H3K4 methylation via oscillatory intracellular cAMP. Activation of Gs-coupled receptors caused a rapid decrease of H3K4me3 by elevating cAMP, whereas stimulation of Gi-coupled receptors increased H3K4me3 by diminishing cAMP. H3K4me3 gradually recovered towards baseline levels after the removal of GPCR ligands, indicating that H3K4me3 oscillates in tandem with GPCR activation. cAMP increased intracellular labile Fe(II), the cofactor for histone demethylases, through a non-canonical cAMP target-Rap guanine nucleotide exchange factor-2 (RapGEF2), which subsequently enhanced endosome acidification and Fe(II) release from the endosome via vacuolar H+-ATPase assembly. Removing Fe(III) from the media blocked intracellular Fe(II) elevation after stimulation of Gs-coupled receptors. Iron chelators and inhibition of KDM5 demethylases abolished cAMP-mediated H3K4me3 demethylation. Taken together, these results suggest a novel function of cAMP signaling in modulating histone demethylation through labile Fe(II)