Extracellular vesicles (EVs) are nano-sized, membrane-bound structures secreted by cells and play critical roles in mediating intercellular signaling. EVs based on their size as well as mechanisms of biosynthesis are categorized as either microvesicles (200–1000 nm) or exosomes (30–200 nm). The EVs carry several biomolecules like proteins, DNAs, RNAs, and lipids into other cells and modulate several cellular functions. Being of very small sizes, it is very challenging to analyze them using conventional microscopes. Here, we report a new method developed by us for visualizing EVs using simple immune-fluorescence based technique, wherein the isolated EVs can be stained with fluorescently tagged antibodies to proteins present in EVs. The stained EVs can then be analyzed by using either confocal or super-resolution microscopes. Our method detailed here is equally effective in staining proteins that are present inside the EVs as well as those localized to the membranes of vesicles. By employing unique staining strategies, we have minimized the background noise and thereby improved the signal strength in confocal microscope. Using electron microscopy, we have ascertained that the structural integrity of the labeled EVs is intact. More importantly, the labeling of EVs does not affect their functionality and their localization can be tracked after its uptake by recipient cells without resorting to any conventional reporter-based strategies or lipophilic dyes. In conclusion, the method described here is a simple, sensitive and efficient immune-fluorescence based method for visualization of molecules within the EVs.

BackgroundNematodes are used in many different fields of science, including environmental and biomedical research. Counting and/or estimating nematode numbers is required during research. Although being one of the most common procedures, this apparently simple task is a time-consuming process, prone to errors and concerns regarding procedure, reliability, and accuracy. When an estimate is necessary, there is a traditional manual counting procedure that in this study it will be called as “drop method” (DM). This popular method that extrapolates an animal count from a small drop of fluid shows a high coefficient of variation. To solve this problem, the present study used the free-living nematode Caenorhabditis elegans to develop a new estimation procedure that was based on a relationship between area and volume of a larger sample.ResultsThe new method showed a low coefficient of variation and a close relationship between estimated and real counts of the total number of nematodes in large C. elegans suspensions. Reactive oxygen concentration was measured as an example of method application and to allow comparison between methods.ConclusionThe proposed method is accurate, facile and reproducible, requiring simple, inexpensive materials that make it an excellent alternative to the DM manual counting procedure. Although the DM is faster, its estimates are not as accurate or as precise as those of the new proposed method.

Tocotrienols (T3) have been shown to represent a very important part of the vitamin E family since they have opened new opportunities to prevent or treat a multitude of age-related chronic diseases. The beneficial effects of T3 include the amelioration of lipid profile, the promotion of Nrf2 mediated cytoprotective activity and the suppression of inflammation. All these effects may be the consequence of the ability of T3 to target multiple pathways. We here propose that these effects may be the result of a single target of T3, namely senescent cells. Indeed, T3 may act by a direct suppression of the senescence-associated secretory phenotype (SASP) produced by senescent cells, mediated by inhibition of NF-kB and mTOR, or may potentially remove the origin of the SASP trough senolysis (selective death of senescent cells). Further studies addressed to investigate the impact of T3 on cellular senescence “in vitro” as well as in experimental models of age-related diseases “in vivo” are clearly encouraged.

BackgroundTo evaluate the effect of qi-nourishing essence-replenishing Chinese herbal medicine combined with chemotherapy in survival of advanced non-small-cell lung cancer(NSCLC) patients with essence and qi deficiency.MethodsA prospective multi-centered randomized controlled study was conducted, and 266 advanced NSCLC patients were enrolled. 126 patients in control group received Vinorelbine plus cisplatin(NP) chemotherapy combined with symptom-oriented Chinese herbs medication(without qi-nourishing essence-replenishing herbs);140 patients in experimental group received NP chemotherapy combined with qi-nourishing essence-replenishing Chinese herbal medication(Kangliu Zengxiao Decoction and modified Feiyanning Decoction, during and after chemotherapy respectively).ResultsOne patient in control and 2 in experimental group were excluded for failure to complete two cycles of chemotherapy. During follow-up, 17 and 7 patients in control and experimental group were excluded respectively(4 and 4 for taking Gefetinib after disease progression, 4 and 2 for receiving other chemotherapeutic regimens, 9 and 1 for lost to follow-up). 239 patients were included in the final analysis (131 in experimental group and 108 in control). Median overall survival in experimental group was significantly longer than control group (14.87vs.12.97 months,P = 0.027). In experimental and control group, 1-year, 3-year, 5-year, 7-year, and 9-year survival rates were 57% vs. 53%, 17% vs. 8%, 10% vs. 2%, 6% vs. 0%, and 6% vs. 0%, respectively.ConclusionQi-nourishing essence-replenishing Chinese herbal medicine combined with chemotherapy improves survival of advanced NSCLC patients with essence and qi deficiency.

BackgroundSubcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method.ResultsThe mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs.ConclusionsThis high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.

BackgroundImmunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.MethodsHere, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost).ResultsThe protocol has been used to generate multiple variants of tetra- and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (Aβ) aggregates in vivo.ConclusionsThis protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.

BackgroundPrimordial germ cells (PGC) are the precursors of the gametes. During pre-natal development, PGC undergo an epigenetic reprogramming when bulk DNA demethylation occurs and is followed by sex-specific de novo methylation. The de novo methylation and the maintenance of the methylation patterns depend on DNA methyltransferases (DNMTs). PGC reprogramming has been widely studied in mice but not in rats. We have previously shown that the rat might be an interesting model to study germ cell development. In face of the difficulties of getting enough PGC for molecular studies, the aim of this study was to propose an alternative method to study rat PGC DNA methylation. Rat embryos were collected at 14, 15 and 19 days post-coitus (dpc) for the analysis of 5mC, 5hmC, DNMT1, DNMT3a and DNMT3b expression or at 16dpc for treatment 5-Aza-CdR, a DNMT inhibitor, in vitro.MethodsOnce collected, the gonads were placed in 24-well plates previously containing 45μm pore membrane and medium with or without 5-Aza-CdR. The culture was kept for five days and medium was changed daily. The gonads were either fixed or submitted to RNA extraction.Results5mC and DNMTs labelling suggests that PGC are undergoing epigenetic reprogramming around 14/15dpc. The in vitro treatment of rat embryonic gonads with 1 μM of 5-Aza-CdR lead to a loss of 5mC labelling and to the activation of Pax6 expression in PGC, but not in somatic cells, suggesting that 5-Aza-CdR promoted a PGC-specific global DNA hypomethylation.ConclusionsThis study suggests that the protocol used here can be a potential method to study the wide DNA demethylation that takes place during PGC reprogramming.

BackgroundThe immunoprecipitation (IP) assay is a valuable molecular biology tool applied across a breadth of fields. The standard assay couples IP to immunoblotting (IP/IB), a procedure severely limited as it is not easily scaled for high-throughput analysis.ResultsHere we describe and characterize a new methodology for fast and reliable evaluation of an immunoprecipitation reaction. FLIP (FLuorescence IP) relies on the expression of the target protein as a chromophore-tagged protein and couples IP with the measurement of fluorescent signal coating agarose beads. We show here that FLIP displays similar sensitivity to the standard IP/IB procedure but is amenable to high-throughput analysis. We applied FLIP to the screening of mouse monoclonal antibodies of unknown behavior in IP procedures. The parallel analysis of the considered antibodies using FLIP and IP/western shows good correlation between the two procedures. We also show application of FLIP using unpurified antibodies (hybridoma supernatant) and we developed a publicly available tool for the easy analysis and quantification of FLIP signals.ConclusionsAltogether, our characterizations of this new methodology show that FLIP is an appealing and reliable tool for any application of high-throughput IP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-016-0046-x) contains supplementary material, which is available to authorized users.

BackgroundIn vitro investigations of membrane proteins usually depend on detergents for protein solubilisation and stabilisation. The amount of detergent bound to a membrane protein is relevant to successful experiment design and data analysis but is often unknown. Triple-detection size-exclusion chromatography enables simultaneous separation of protein/detergent complexes and protein-free detergent micelles and determination of their molar masses in a straightforward and absolute manner. Size-exclusion chromatography is used to separate different species, while ultraviolet absorbance, static light scattering, and refractive index measurements allow molar mass determination of protein and detergent components.ResultsWe refined standard experimental and data-analysis procedures for challenging membrane-protein samples that elude routine approaches. The general procedures including preparatory steps, measurements, and data analysis for the characterisation of both routine and complex samples in difficult solvents such as concentrated denaturant solutions are demonstrated. The applicability of the protocol but also its limitations and possible solutions are discussed, and an extensive troubleshooting section is provided.ConclusionsWe established and validated a protocol for triple-detection size-exclusion chromatography that enables the inexperienced user to perform and analyse measurements of well-behaved protein/detergent complexes. More experienced users are provided with an example of a more sophisticated analysis procedure allowing mass determination under challenging separation conditions.