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21.
The decision to use 10% neutral buffered formalin fixed, paraffin embedded (FFPE) archival pathology material may be dictated by the cancer research question or analytical technique, or may be governed by national ethical, legal and social implications (ELSI), biobank, and sample availability and access policy. Biobanked samples of common tumors are likely to be available, but not all samples will be annotated with treatment and outcomes data and this may limit their application. Tumors that are rare or very small exist mostly in FFPE pathology archives. Pathology departments worldwide contain millions of FFPE archival samples, but there are challenges to availability. Pathology departments lack resources for retrieving materials for research or for having pathologists select precise areas in paraffin blocks, a critical quality control step. When samples must be sourced from several pathology departments, different fixation and tissue processing approaches create variability in quality. Researchers must decide what sample quality and quality tolerance fit their specific purpose and whether sample enrichment is required. Recent publications report variable success with techniques modified to examine all common species of molecular targets in FFPE samples. Rigorous quality management may be particularly important in sample preparation for next generation sequencing and for optimizing the quality of extracted proteins for proteomics studies. Unpredictable failures, including unpublished ones, likely are related to pre-analytical factors, unstable molecular targets, biological and clinical sampling factors associated with specific tissue types or suboptimal quality management of pathology archives. Reproducible results depend on adherence to pre-analytical phase standards for molecular in vitro diagnostic analyses for DNA, RNA and in particular, extracted proteins. With continuing adaptations of techniques for application to FFPE, the potential to acquire much larger numbers of FFPE samples and the greater convenience of using FFPE in assays for precision medicine, the choice of material in the future will become increasingly biased toward FFPE samples from pathology archives. Recognition that FFPE samples may harbor greater variation in quality than frozen samples for several reasons, including variations in fixation and tissue processing, requires that FFPE results be validated provided a cohort of frozen tissue samples is available. 相似文献
22.
Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans. 相似文献
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Lin C Zhong Z Lok MC Jiang X Hennink WE Feijen J Engbersen JF 《Bioconjugate chemistry》2007,18(1):138-145
A series of novel bioreducible poly(amido amine)s containing multiple disulfide linkages (SS-PAAs) were synthesized and evaluated as nonviral gene vectors. These linear SS-PAAs could be easily obtained by Michael-type polyaddition of various primary amines to the disulfide-containing cystamine bisacrylamide. The SS-PAA polymers are relatively stable in medium mimicking physiological conditions (pH 7.4, 150 mM PBS, 37 degrees C), but are rapidly degraded in the presence of 2.5 mM DTT, mimicking the intracellular reductive environment (pH 7.4, [R-SH] = 5 mM, 37 degrees C). The polymers efficiently condense DNA into nanoscaled (<200 nm) and positively charged (>+20 mV) polyplexes that are stable under neutral conditions but are rapidly destabilized in a reductive environment, as was revealed by both dynamic light scatting measurement and agarose gel assays. Moreover, most of the poly(amido amine)s possess buffer capacities in the pH range pH 7.4-5.1 that are even higher than polyethylenimine (pEI), a property that may favorably contribute to the endosomal escape of the polyplexes. Polyplexes of four of the seven SS-PAAs studied were able to transfect COS-7 cells in vitro with transfection efficiencies significantly higher than those of branched pEI, being one of the most effective polymeric gene carriers reported to date. Importantly, also in the presence of serum, a high level of gene expression could be observed when the incubation time was elongated from 1 h to 4 h. XTT assays showed that SS-PAAs and their polyplexes possess essentially no or only very low cytotoxicity at concentrations where the highest transfection activity is observed. The results indicate that bioreducible poly(amido amine)s have excellent properties for the development of highly potent and nontoxic polymeric gene carriers. 相似文献
25.
In this study we describe the sociodemographic characteristics of people participating in a clinical trial on the safety and immunogenicity of a H5N1 influenza vaccine and we identify the main motivations for joining it. 相似文献
26.
We have identified a novel N -acetylgalactosaminyltransferase activity in
lactating bovine mammary gland membranes. Acceptor specificity studies and
analysis of products obtained in vitro by 400 MHz1H-NMR spectroscopy
revealed that the enzyme catalyses the transfer of N - acetylgalactosamine
(GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal,
beta-linked N -acetylglucosamine (GlcNAc) residue and establishes a
beta1-->4-linkage forming a GalNAcbeta1-->4GlcNAc ( N, N
'-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be
identified as a UDP-GalNAc:GlcNAcbeta-R beta1-->4-N-
acetylgalactosaminyltransferase (beta4-GalNAcT). This enzyme resembles
invertebrate beta4-GalNAcT as well as mammalian beta4-
galactosyltransferase (beta4-GalT) in acceptor specificity. It can,
however, be clearly distinguished from the pituitary hormone-specific
beta4-GalNAcT by its incapability of acting with an elevated activity on a
glycoprotein substrate carrying a hormone-specific peptide motif.
Furthermore, the GalNAcT activity appeared not to be due to a promiscuous
action of a beta4-GalT as could be demonstrated by comparing the
beta4-GalNAcT and beta4-GalT activities of the mammary gland, bovine
colostrum, and purified beta4-GalT, by competition studies with UDP-GalNAc
and UDP-Gal, and by use of an anti-beta4-GalT polyclonal inhibiting
antibody. Interestingly, under conditions where mammalian beta4-GalT forms
with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary
gland beta4-GalNAcT was similarly induced by alpha-LA to act on Glc with an
increased efficiency yielding the lactose analog GalNAcbeta1-->4Glc.
This enzyme thus forms the second example of a mammalian
glycosyltransferase the specificity of which can be modified by this milk
protein. It is proposed that the mammary gland beta4-GalNAcT functions in
the synthesis of lacdiNAc- based, complex-type glycans frequently occurring
on bovine milk glycoproteins. The action of this enzyme is to be considered
when aiming at the production of properly glycosylated protein
biopharmaceuticals in the milk of transgenic dairy animals.
相似文献
27.
Sultmann H; Mayer WE; Figueroa F; Tichy H; Klein J 《Molecular biology and evolution》1995,12(6):1033-1047
The recent explosive adaptive radiation of cichlids in the great lakes of
Africa has attracted the attention of both morphologists and molecular
biologists. To decipher the phylogenetic relationships among the various
taxa within the family Cichlidae is a prerequisite for answering some
fundamental questions about the nature of the speciation process. In the
present study, we used the random amplification of polymorphic DNA (RAPD)
technique to obtain sequence differences between selected cichlid species.
We then designed specific primers based on these sequences and used them to
amplify template DNA from a large number of species by the polymerase chain
reaction (PCR). We sequenced the amplified products and searched the
sequences for indels and shared substitutions. We identified a number of
such characters at three loci-- DXTU1, DXTU2, and DXTU3--and used them for
phylogenetic and cladistic analysis of the relationships among the various
cichlid groups. Our studies assign an outgroup position to Neotropical
cichlids in relation to African cichlids, provide evidence for a
sister-group relationship of tilapiines to the haplochromines, group
Cyphotilapia frontosa with the lamprologines of Lake Tanganyika, place
Astatoreochromis alluaudi to an outgroup position with respect to other
haplochromines of Lakes Victoria and Malawi, and provide additional support
for the monophyly of the remaining Lake Victoria haplochromines and the
Lake Malawi haplochromines. The described approach holds great promise for
further resolution of cichlid phylogeny.
相似文献
28.
Abstract The mating behavior of cat flea, Ctenocepholides felis (Bouche) was studied on an artificial feeding device. Male and female can mate repeatedly with same partner or Merent ones. In the situation of male: female ratio of 1 :5, each mating lasted an average of 6.6 min, with a mean interval between matings at 2.5 min., compared to 11. 1 min and 12.1 min respectively in a cell with 5 males and 1 female. As many as 48 mating events were observed for one male during an 8 h period. One female mated 27 times in 7 h with 5 males in the same cell. Newly emerged males and females can not mate before blood meal and about 24 h blood feeding is rewuired for successful mating. Newly emerged males can not mate with fed females (fed for 48 h), but fed males can mate with newly emerged females who are feeding the blood. Significantly more male contacts and male-male mating attempts were observed after the paper treated with female extract was introduced into the cell. The paper contacts and mating attempts were 16.75–32.25 times and 15.75–31.38 times, respectively, on average during a period of 20 min when different doses (FE) of extract were provided. 相似文献
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Zhong Z Feijen J Lok MC Hennink WE Christensen LV Yockman JW Kim YH Kim SW 《Biomacromolecules》2005,6(6):3440-3448
Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems. 相似文献