KRAS Mutation Detection in Paired Frozen and Formalin-Fixed Paraffin-Embedded (FFPE) Colorectal Cancer Tissues

KRAS mutation has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies of KRAS mutation analysis have been performed using homogenously archived CRC specimens, and studies that compare freshly frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens of CRC are lacking. The aim of the present study was to evaluate the impact of tissue preservation on the determination of KRAS mutational status. A series of 131 mCRC fresh-frozen tissues were first analyzed using both high-resolution melting (HRM) and direct sequencing. KRAS mutations were found in 47/131 (35.8%) using both approaches. Out of the 47 samples that were positive for KRAS mutations, 33 had available matched FFPE specimens. Using HRM, 2/33 (6%) demonstrated suboptimal template amplification, and 2/33 (6%) expressed an erroneous wild-type KRAS profile. Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations. Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping

Accumulation of Endogenous LITAF in Aggresomes

LITAF is a 161 amino acid cellular protein which includes a proline rich N-terminus and a conserved C-terminal domain known as the simple-like domain. Mutations in LITAF have been identified in Charcot-Marie tooth disease, a disease characterized by protein aggregates. Cells transfected with cellular LITAF reveal that LITAF is localized to late endosomes/lysosomes. Here we investigated the intracellular localization of endogenous LITAF. We demonstrated that endogenous LITAF accumulates at a discrete cytoplasmic site in BGMK cells that we identify as the aggresome. To determine the domain within LITAF that is responsible for the localization of LITAF to aggresomes, we created a construct that contained the C-terminal simple-like domain of LITAF and found that this construct also localizes to aggresomes. These data suggest the simple-like domain is responsible for targeting endogenous LITAF to the aggresome

The LSD1-Interacting Protein GILP Is a LITAF Domain Protein That Negatively Regulates Hypersensitive Cell Death in Arabidopsis

Hypersensitive cell death, a form of avirulent pathogen-induced programmed cell death (PCD), is one of the most efficient plant innate immunity. However, its regulatory mechanism is poorly understood. AtLSD1 is an important negative regulator of PCD and only two proteins, AtbZIP10 and AtMC1, have been reported to interact with AtLSD1.To identify a novel regulator of hypersensitive cell death, we investigate the possible role of plant LITAF domain protein GILP in hypersensitive cell death. Subcellular localization analysis showed that AtGILP is localized in the plasma membrane and its plasma membrane localization is dependent on its LITAF domain. Yeast two-hybrid and pull-down assays demonstrated that AtGILP interacts with AtLSD1. Pull-down assays showed that both the N-terminal and the C-terminal domains of AtGILP are sufficient for interactions with AtLSD1 and that the N-terminal domain of AtLSD1 is involved in the interaction with AtGILP. Real-time PCR analysis showed that AtGILP expression is up-regulated by the avirulent pathogen Pseudomonas syringae pv. tomato DC3000 avrRpt2 (Pst avrRpt2) and fumonisin B1 (FB1) that trigger PCD. Compared with wild-type plants, transgenic plants overexpressing AtGILP exhibited significantly less cell death when inoculated with Pst avrRpt2, indicating that AtGILP negatively regulates hypersensitive cell death.These results suggest that the LITAF domain protein AtGILP localizes in the plasma membrane, interacts with AtLSD1, and is involved in negatively regulating PCD. We propose that AtGILP functions as a membrane anchor, bringing other regulators of PCD, such as AtLSD1, to the plasma membrane. Human LITAF domain protein may be involved in the regulation of PCD, suggesting the evolutionarily conserved function of LITAF domain proteins in the regulation of PCD

Traditional knowledge of wild edible plants used in Palestine (Northern West Bank): A comparative study

<p>Abstract</p> <p>Background</p> <p>A comparative food ethnobotanical study was carried out in fifteen local communities distributed in five districts in the Palestinian Authority, PA (northern West Bank), six of which were located in Nablus, two in Jenin, two in Salfit, three in Qalqilia, and two in Tulkarm. These are among the areas in the PA whose rural inhabitants primarily subsisted on agriculture and therefore still preserve the traditional knowledge on wild edible plants.</p> <p>Methods</p> <p>Data on the use of wild edible plants were collected for one-year period, through informed consent semi-structured interviews with 190 local informants. A semi-quantitative approach was used to document use diversity, and relative importance of each species.</p> <p>Results and discussion</p> <p>The study recorded 100 wild edible plant species, seventy six of which were mentioned by three informants and above and were distributed across 70 genera and 26 families. The most significant species include <it>Majorana syriaca, Foeniculum vulgare, Malvasylvestris</it>, <it>Salvia fruticosa, Cyclamen persicum, Micromeria fruticosa, Arum palaestinum, Trigonella foenum-graecum</it>, <it>Gundelia tournefortii</it>, and <it>Matricaria aurea</it>. All the ten species with the highest mean cultural importance values (mCI), were cited in all five areas. Moreover, most were important in every region. A common cultural background may explain these similarities. One taxon (<it>Majoranasyriaca</it>) in particular was found to be among the most quoted species in almost all areas surveyed. CI values, as a measure of traditional botanical knowledge, for edible species in relatively remote and isolated areas (Qalqilia, and Salfit) were generally higher than for the same species in other areas. This can be attributed to the fact that local knowledge of wild edible plants and plant gathering are more spread in remote or isolated areas.</p> <p>Conclusion</p> <p>Gathering, processing and consuming wild edible plants are still practiced in all the studied Palestinian areas. About 26 % (26/100) of the recorded wild botanicals including the most quoted and with highest mCI values, are currently gathered and utilized in all the areas, demonstrating that there are ethnobotanical contact points among the various Palestinian regions. The habit of using wild edible plants is still alive in the PA, but is disappearing. Therefore, the recording, preserving, and infusing of this knowledge to future generations is pressing and fundamental.</p

Improved functionalization of oleic acid-coated iron oxide nanoparticles for biomedical applications

Superparamagnetic iron oxide nanoparticles can providemultiple benefits for biomedical applications in aqueous environments such asmagnetic separation or magnetic resonance imaging. To increase the colloidal stability and allow subsequent reactions, the introduction of hydrophilic functional groups onto the particles’ surface is essential. During this process, the original coating is exchanged by preferably covalently bonded ligands such as trialkoxysilanes. The duration of the silane exchange reaction, which commonly takes more than 24 h, is an important drawback for this approach. In this paper, we present a novel method, which introduces ultrasonication as an energy source to dramatically accelerate this process, resulting in high-quality waterdispersible nanoparticles around 10 nmin size. To prove the generic character, different functional groups were introduced on the surface including polyethylene glycol chains, carboxylic acid, amine, and thiol groups. Their colloidal stability in various aqueous buffer solutions as well as human plasma and serum was investigated to allow implementation in biomedical and sensing applications.status: publishe

Nodule-specific cysteine-rich peptide 343 is required for symbiotic nitrogen fixation in Medicago truncatula

Symbiotic interactions between legumes and rhizobia lead to the development of root nodules and nitrogen fixation by differentiated bacteroids within nodules. Differentiation of the endosymbionts is reversible or terminal, determined by plant effectors. In inverted repeat lacking clade legumes, nodule-specific cysteine-rich (NCR) peptides control the terminal differentiation of bacteroids. Medicago truncatula contains ∼700 NCR-coding genes. However, the role of few NCR peptides has been demonstrated. Here, we report characterization of fast neutron 2106 (FN2106), a symbiotic nitrogen fixation defective (fix−) mutant of M. truncatula. Using a transcript-based approach, together with linkage and complementation tests, we showed that loss-of-function of NCR343 results in impaired bacteroid differentiation and/or maintenance and premature nodule senescence of the FN2106 mutant. NCR343 was specifically expressed in nodules. Subcellular localization studies showed that the functional NCR343-YFP fusion protein colocalizes with bacteroids in symbiosomes in infected nodule cells. Transcriptomic analyses identified senescence-, but not defense-related genes, as being significantly upregulated in ncr343 (FN2106) nodules. Taken together, results from our phenotypic and transcriptomic analyses of a loss-of-function ncr343 mutant demonstrate an essential role of NCR343 in bacteroid differentiation and/or maintenance required for symbiotic nitrogen fixation