Effects of neo-adjuvant chemotherapy for oesophago-gastric cancer on neuro-muscular gastric function

Delayed gastric emptying symptoms are often reported after chemotherapy. This study aims to characterise the effects of chemotherapy on gastric neuro-muscular function. Patients undergoing elective surgery for oesophago-gastric cancer were recruited. Acetylcholinesterase, nNOS, ghrelin receptor and motilin expressions were studied in gastric sections from patients receiving no chemotherapy (n = 3) or oesophageal (n = 2) or gastric (n = 2) chemotherapy. A scoring system quantified staining intensity (0–3; no staining to strong). Stomach sections were separately suspended in tissue baths for electrical field stimulation (EFS) and exposure to erythromycin or carbachol; three patients had no chemotherapy; four completed cisplatin-based chemotherapy within 6 weeks prior to surgery. AChE expression was markedly decreased after chemotherapy (scores 2.3 ± 0.7, 0.5 ± 0.2 and 0 ± 0 in non-chemotherapy, oesophageal- and gastric-chemotherapy groups (p < 0.03 each) respectively. Ghrelin receptor and motilin expression tended to increase (ghrelin: 0.7 ± 0.4 vs 2.0 ± 0.4 and 1.2 ± 0.2 respectively; p = 0.04 and p = 0.2; motilin: 0.7 ± 0.5 vs 2.2 ± 0.5 and 2.0 ± 0.7; p = 0.06 and p = 0.16). Maximal contraction to carbachol was 3.7 ± 0.7 g and 1.9 ± 0.8 g (longitudinal muscle) and 3.4 ± 0.4 g and 1.6 ± 0.6 (circular) in non-chemotherapy and chemotherapy tissues respectively (p < 0.05 each). There were loss of AChE and reduction in contractility to carbachol. The tendency for ghrelin receptors to increase suggests an attempt to upregulate compensating systems. Our study offers a mechanism by which chemotherapy markedly alters neuro-muscular gastric function

siRNA-Like Double-Stranded RNAs Are Specifically Protected Against Degradation in Human Cell Extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time

A recent bottleneck of Y chromosome diversity coincides with a global change in culture

Contains fulltext : 153022.pdf (publisher's version ) (Open Access)It is commonly thought that human genetic diversity in non-African populations was shaped primarily by an out-of-Africa dispersal 50-100 thousand yr ago (kya). Here, we present a study of 456 geographically diverse high-coverage Y chromosome sequences, including 299 newly reported samples. Applying ancient DNA calibration, we date the Y-chromosomal most recent common ancestor (MRCA) in Africa at 254 (95% CI 192-307) kya and detect a cluster of major non-African founder haplogroups in a narrow time interval at 47-52 kya, consistent with a rapid initial colonization model of Eurasia and Oceania after the out-of-Africa bottleneck. In contrast to demographic reconstructions based on mtDNA, we infer a second strong bottleneck in Y-chromosome lineages dating to the last 10 ky. We hypothesize that this bottleneck is caused by cultural changes affecting variance of reproductive success among males

RNA “Traffic Lights”: An Analytical Tool to Monitor siRNA Integrity

The combination of thiazole orange and thiazole red as an internal energy transfer-based fluorophore pair in oligonucleotides provides an outstanding analytical tool to follow DNA/RNA hybridization through a distinct fluorescence color change from red to green. Herein, we demonstrate that this concept can be applied to small interfering RNA (siRNA) to monitor RNA integrity in living cells in real time with a remarkable dynamic range and excellent contrast ratios in cellular media. Furthermore, we show that our siRNA-sensors still possess their gene silencing function toward the knockdown of enhanced green fluorescent protein in CHO-K1 cells

Upconversion nanoparticle-based FRET system for study of siRNA in live cells

10.1021/la904011qLangmuir2696689-6694LANG

FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes

The assembly, stability, and timely disassembly of short interfering RNA (siRNA) nanocomplexes have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or their environment may facilitate the study and further development of new siRNA nanocomplexes. Herein, we study Förster resonance energy transfer (FRET)-labeled siRNA probes that can track the assembly, stability, and disassembly of siRNA nanocomplexes in different environments. The probe is composed of two identical siRNAs, each labeled with a fluorophore. Upon nanocomplex formation, the siRNA-bound fluorophores become locally aggregated within the nanocomplex and undergo FRET. A key advantage of this technique is that the delivery vehicle (DV) need not be labeled, thus enabling the characterization of a large variety of nanocarriers, some of which may be difficult or even impossible to label. We demonstrate proof-of-concept by measuring the assembly of various DVs with siRNAs and show good agreement with gel electrophoresis experiments. As a consequence of not having to label the DV, we are able to determine nanocomplex biophysical parameters such as the extracellular apparent dissociation constants (KD) and intracellular disassembly half-life for several in-house and proprietary commercial DVs. Furthermore, the lack of DV modification allows for a true direct comparison between DVs as well as correlation between their biophysical properties and gene silencing.National Institutes of Health (U.S.) (NIH grant R37-EB000244)National Institutes of Health (U.S.) (NIH grant R01-CA132091)National Institutes of Health (U.S.) (NIH grant R01-CA115527)National Institutes of Health (U.S.) (Postdoctoral Fellowship

Localization of Double-stranded Small Interfering RNA to Cytoplasmic Processing Bodies Is Ago2 Dependent and Results in Up-Regulation of GW182 and Argonaute-2

Processing bodies (P-bodies) are cytoplasmic foci implicated in the regulation of mRNA translation, storage, and degradation. Key effectors of microRNA (miRNA)-mediated RNA interference (RNAi), such as Argonaute-2 (Ago2), miRNAs, and their cognate mRNAs, are localized to these structures; however, the precise role that P-bodies and their component proteins play in small interfering RNA (siRNA)-mediated RNAi remains unclear. Here, we investigate the relationship between siRNA-mediated RNAi, RNAi machinery proteins, and P-bodies. We show that upon transfection into cells, siRNAs rapidly localize to P-bodies in their native double-stranded conformation, as indicated by fluorescence resonance energy transfer imaging and that Ago2 is at least in part responsible for this siRNA localization pattern, indicating RISC involvement. Furthermore, siRNA transfection induces up-regulated expression of both GW182, a key P-body component, and Ago2, indicating that P-body localization and interaction with GW182 and Ago2 are important in siRNA-mediated RNAi. By virtue of being centers where these proteins and siRNAs aggregate, we propose that the P-body microenvironment, whether as microscopically visible foci or submicroscopic protein complexes, facilitates siRNA processing and siRNA-mediated silencing through the action of its component proteins