The Synthesis and Biological Assessment of Novel Ursolic Acid Derivatives as Potential Chemotherapeutic Agents for Cancer

Glioblastoma (GBM) is considered to be the most biologically aggressive type of brain tumour accounting for approximately 48% of all malignant primary brain tumours. GBM patients diagnosed have poor prognosis with a low five-year survival rate of The aim of this study was to develop novel UA derivatives to enhance its bioavailability. Nine novel UA derivatives: three different diamine linkers, with Boc-protected and deprotected ends, and with folic acid were designed and synthesized to improve compound activity and/or delivery. The structures of the newly synthesised compounds were confirmed using mass spectrometry, FTIR, 1H NMR and 13C NMR. The structural activity relationship (SAR) of UA and novel UA derivatives that were designed to improve its activity and bioavailability (predicted ADMET profile) were explored, and molecular docking studies against proposed targets – FOLR1 and/or VRK1 were utilised, with an in-depth analysis of predicted interactions. The cytotoxic activity was determined using alamarBlueTM cell viability assay in a panel of cancer cell lines and normal cells. It was found that the conjugation of folic acid to UA decreased its cytotoxic activity. Interestingly, our study showed that the Boc-protected compounds have delayed cytotoxicity in comparison to deprotected compounds, which are more evident in U-251 MG and A431 cell lines using 2D cell culture assay. Whereas in 3D cell culture, only deprotected compounds exhibited an activity. The deprotected novel UA derivatives also retained the inherent anti-proliferative and anti-migratory effect of UA in U-251 MG cells. The synergistic studies focusing on the cell membrane damage performed showed that novel UA derivatives (7 – 12) may have a protective effect when exposed to radioactivity. In addition, the initial inhibitor studies suggests that compounds 8 and 10 at longer timepoint, may trigger multiple cell demise pathway. Based on these preliminary results, this study provides a new insight into using novel UA derivatives and a possible MOA for their anti-cancer effect

Ultrasound 96 Probe Device Protocol for cancer cell treatment

Ultrasound is a sound wave with frequencies ranging between 20 kHz and 20 MHz. Ultrasound is able to temporarily and repeatedly open the BBB safely and enhance chemotherapeutic delivery without adverse effects. This novel technique in drug delivery benefits from the powerful ability of ultrasound to produce cavitation activity. Cavitation is the generation and activity of gas-filled bubbles in a medium exposed to ultrasound. As the pressure wave passes through the media, gas bubbles expand at low pressure and contract at high pressure. This leads to oscillation which produces a circulating fluid flow known as microstreaming around the bubble with velocities and shear rates proportional to the amplitude of the oscillation. At high amplitudes the associated shear forces can cut open liposome

Whole genome transcript profiling from fingerstick blood samples: a comparison and feasibility study

<p>Abstract</p> <p>Background</p> <p>Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5 mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small human samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 μl of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit.</p> <p>Results</p> <p>From two sets of fingerstick collections, about 70 uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6 ng total RNA with an average RIN of 9.3. The post-amplification yields for 50 ng of total RNA averaged at 7.0 ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with r²values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r²values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.94 to 0.96 for fingerstick collection 2.</p> <p>Conclusions</p> <p>Our comparisons of RNA quality and gene expression data of the fingerstick method with traditionally processed sample workflows demonstrate excellent RNA quality from the capillary collection as well as very high correlations of gene expression data.</p

Valorisation of Phytochemical from Sitka Spruce (Picea Sitchensis) Needles: Impact of Ultrasound/microwave-assisted Extraction

Sitka spruce (Picea sitchensis) needles contain a variety of bioactive compounds including phenolic compounds and flavonoids, many of which have been used in the cosmetic, pharmaceutical, and food industries. This study aimed to investigate the effects of novel extraction techniques, including ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and simultaneous ultrasound–microwave-assisted extraction (UMAE) on the recovery of phenolic, flavonoids and associated antioxidant and anti-cancer properties from Sitka spruce (Picea sitchensis) needles. The ferric reducing antioxidant power (FRAP) assay was used to evaluate the antioxidant capacity, and the Alamar Blue assay using the human brain glioblastoma cancer cell line (U-251 MG) was used to evaluate the cytotoxicity activity. Results showed that US-probe accomplished the highest recovery of phenolic and flavonoids at 38 W cm−2 for 10 min (106.3 ± 2.5 mg GAE g−1 DW and 63.2 ± 3.8 mg QE g−1 DW, respectively). Hence, the highest cytotoxicity activity of IC50 (0.0114% w/v) was achieved by US-probe at 19 W cm−2 for 10 min. However, the antioxidant capacity of (2591.3 ± 92.5 mM TE g−1 DW) was achieved under UMAE at ultrasound intensity of 38 W cm−2, microwave power of 302.4 W for 10 min. This study emphasised the potential application of UAE and MAE in the extraction of bioactive as an environmentally friendly method to be used in the valorisation of by-products in food and agro-industries. This supports the use of renewable natural resources in an efficient way to produce high-value compounds therefore it is in line with the new era of bioeconomy and its new biorefinery concepts

Ursolic Acid Inhibits Collective Cell Migration and Promotes JNK-Dependent Lysosomal Associated Cell Death in Gioblastoma Multiforme Cells

Ursolic acid (UA) is a bioactive compound which has demonstrated therapeutic efficacy in a variety of cancer cell lines. UA activates various signalling pathways in Glioblastoma multiforme (GBM) and offers a promising starting point in drug discovery; however, understanding the relationship between cell death and migration has yet to be elucidated. UA induces a dose dependent cytotoxic response demonstrated by flow cytometry and biochemical cytotoxicity assays. Inhibitor and fluorescent probe studies demonstrate that UA induces a caspase independent, JNK dependent, mechanism of cell death. Migration studies established that UA inhibits GBM collective cell migration in a time dependent manner that is independent of the JNK signalling pathway. Cytotoxicity induced by UA results in the formation of acidic vesicle organelles (AVOs), speculating the activation of autophagy. However, inhibitor and spectrophotometric analysis demonstrated that autophagy was not responsible for the formation of the AVOs. Confocal microscopy and isosurface visualisation determined co-localisation of lysosomes with the previously identified AVOs, thus providing evidence that lysosomes are likely to be playing a role in UA induced cell death. Collectively, our data identify that UA rapidly induces a lysosomal associated mechanism of cell death in addition to UA acting as an inhibitor of GBM collective cell migration

Influence of Molecular Weight Fractionation on the Antimicrobial and Anticancer Properties of a Fucoidan Rich-Extract From The Macroalgae Fucus Vesiculosus

The objective of this study was to investigate the antimicrobial and anticancer properties of a fucoidan extract and subsequent fractions isolated from the macroalgae Fucus vesiculosus. The fractions obtained (\u3e300 kDa,kDa,kDa,kDa) could inhibit the growth of B. subtilis, E. coli, L. innocua and P. fluorescens when assayed at concentrations between 12,500 and 25,000 ppm. The bacterial growth was monitored by optical density (OD) measurements (600 nm, 24 h) at 30 °C or 37 °C, depending upon on the strain used. The extracted fractions were also tested for cytotoxicity against brain glioblastoma cancer cells using the Alamar Blue assay for 24 h, 48 h and 6 days. The \u3e300 kDa fraction presented the lowest IC50 values (0.052% - 24 h; 0.032% - 6 days). The potential bioactivity of fucoidan as an antimicrobial and anticancer agent was demonstrated in this study. Hence, the related mechanisms of action should be explored in a near future

Gene Expression in Biopsies of Acute Rejection and Interstitial Fibrosis/Tubular Atrophy Reveals Highly Shared Mechanisms That Correlate With Worse Long‐Term Outcomes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/1/ajt13728.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/2/ajt13728-sup-0005-AppendixS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/3/ajt13728-sup-0003-AppendixS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/4/ajt13728_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/5/ajt13728-sup-0004-AppendixS4.pd

Differential sensitivity of target genes to translational repression by miR-17~92

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genesCX is a Pew Scholar in Biomedical Sciences. This study is supported by the PEW Charitable Trusts, Cancer Research Institute, National Institute of Health (R01AI087634, R01AI089854, RC1CA146299, R56AI110403, and R01AI121155 to CX), National Natural Science Foundation of China (31570882 to WHL, 31570883 to NX, 31570911 to GF, 91429301 to JH, 31671428 and 31500665 to YZ), 1000 Young Talents Program of China (K08008 to NX), 100 Talents Program of The Chinese Academy of Sciences (YZ), National Program on Key Basic Research Project of China (2016YFA0501900 to YZ), the Fundamental Research Funds for the Central Universities of China (20720150065 to NX and GF), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A1A01052387 to SGK, NRF-2016R1A4A1010115 to SGK and PHK), and 2016 Research Grant from Kangwon National University (SGK)

Biomarkers for Early and Late Stage Chronic Allograft Nephropathy by Proteogenomic Profiling of Peripheral Blood

Despite significant improvements in life expectancy of kidney transplant patients due to advances in surgery and immunosuppression, Chronic Allograft Nephropathy (CAN) remains a daunting problem. A complex network of cellular mechanisms in both graft and peripheral immune compartments complicates the non-invasive diagnosis of CAN, which still requires biopsy histology. This is compounded by non-immunological factors contributing to graft injury. There is a pressing need to identify and validate minimally invasive biomarkers for CAN to serve as early predictors of graft loss and as metrics for managing long-term immunosuppression.We used DNA microarrays, tandem mass spectroscopy proteomics and bioinformatics to identify genomic and proteomic markers of mild and moderate/severe CAN in peripheral blood of two distinct cohorts (n = 77 total) of kidney transplant patients with biopsy-documented histology.Gene expression profiles reveal over 2400 genes for mild CAN, and over 700 for moderate/severe CAN. A consensus analysis reveals 393 (mild) and 63 (moderate/severe) final candidates as CAN markers with predictive accuracy of 80% (mild) and 92% (moderate/severe). Proteomic profiles show over 500 candidates each, for both stages of CAN including 302 proteins unique to mild and 509 unique to moderate/severe CAN.This study identifies several unique signatures of transcript and protein biomarkers with high predictive accuracies for mild and moderate/severe CAN, the most common cause of late allograft failure. These biomarkers are the necessary first step to a proteogenomic classification of CAN based on peripheral blood profiling and will be the targets of a prospective clinical validation study