30,190 research outputs found
TREATMENT OF HEMOPHILIA WITH HUMAN FACTORIX PRODUCED IN MAMIMARY TISSUE OF TRANSGENIC MAMMALS
Recombinant Factor IX characterized by a high percentage of active protein can be obtained in the milk of transgenic animals that incorporate chimeric DNA molecules according to the present invention. Transgenic animals of the present invention are produced by introducing into developing embryos DNA that encodes Factor IX, such that the foreign DNA is stably incorporated in the DNA of germ line cells of the mature animal. Particularly efficient expression was accomplished using a chimeric construct comprising a mammary gland specific promoter, Factor IX cDNA that lacked the complete or any portion of the 5\u27-untranslated and 3\u27-un-translated region, which is substituted with a 5- and 3\u27-end of the mouse whey acidic protein gene. In vitro cell cultures of cells explanted from the transgenic mammal of the invention and methods of producing Factor IX from such said culture and methods of treating hemophilia B are also described
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A Label-Free Platform for Identification of Exosomes from Different Sources.
Exosomes contain cell- and cell-state-specific cargos of proteins, lipids, and nucleic acids and play significant roles in cell signaling and cell-cell communication. Current research into exosome-based biomarkers has relied largely on analyzing candidate biomarkers, i.e., specific proteins or nucleic acids. However, this approach may miss important biomarkers that are yet to be identified. Alternative approaches are to analyze the entire exosome system, either by "omics" methods or by techniques that provide "fingerprints" of the system without identifying each individual biomolecule component. Here, we describe a platform of the latter type, which is based on surface-enhanced Raman spectroscopy (SERS) in combination with multivariate analysis, and demonstrate the utility of this platform for analyzing exosomes derived from different biological sources. First, we examined whether this analysis could use exosomes isolated from fetal bovine serum using a simple, commercially available isolation kit or necessitates the higher purity achieved by the "gold standard" ultracentrifugation/filtration procedure. Our data demonstrate that the latter method is required for this type of analysis. Having established this requirement, we rigorously analyzed the Raman spectral signature of individual exosomes using a unique, hybrid SERS substrate made of a graphene-covered Au surface containing a quasi-periodic array of pyramids. To examine the source of the Raman signal, we used Raman mapping of low and high spatial resolution combined with morphological identification of exosomes by scanning electron microscopy. Both approaches suggested that the spectra were collected from single exosomes. Finally, we demonstrate for the first time that our platform can distinguish among exosomes from different biological sources based on their Raman signature, a promising approach for developing exosome-based fingerprinting. Our study serves as a solid technological foundation for future exploration of the roles of exosomes in various biological processes and their use as biomarkers for disease diagnosis and treatment monitoring
Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase
A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis—an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.National Institutes of Health (U.S.) (Grant R01CA103866)National Institutes of Health (U.S.) (Grant R37AI047389
HISTOLOGICAL STUDIES OF BREWERY SPENT GRAINS IN DIETARY PROTEIN FORMULATION IN DONRYU RATS
The increasing production of large tonnage of products in brewing industries continually generates lots of solid waste
which includes spent grains, surplus yeast, malt sprout and cullet. The disposal of spent grains is often a problem and
poses major health and environmental challenges, thereby making it imminently necessary to explore alternatives for its
management. This paper focuses on investigating the effects of Brewery Spent Grain formulated diet on haematological,
biochemical, histological and growth performance of Donryu rats. The rats were allocated into six dietary treatment
groups and fed on a short-term study with diet containing graded levels of spent grains from 0, 3, 6, 9, 12 and 100%
weight/weight. The outcome demonstrated that formulated diet had a positive effect on the growth performance of the
rats up to levels of 6% inclusions, while the haematological and biochemical evaluation revealed that threshold limit
should not exceed 9% of the grain. However, the histological study on the liver indicated a limit of 3% inclusion in feed
without serious adverse effect. Thus invariably showing that blend between ranges 1-3% is appropriate for the utilization
of the waste in human food without adverse effect on the liver organ. The economic advantage accruing from this waste
conversion process not only solves problem of waste disposal but also handle issues of malnutrition in feeding ration
NMR for sample quality assessment in metabolomics.
Abstract The EU Framework 7 project SPIDIA was the occasion for development of NMR approaches to evaluate the impact of different pre-analytical treatments on the quality of biological samples dedicated to metabolomics. Systematic simulation of different pre-analytical procedures was performed on urine and blood serum and plasma. Here we review the key aspects of these studies that have led to the development of CEN technical specifications, to be translated into ISO/IS in the course of the EU Horizon 2020 project SPIDIA4P. Inspired by the SPIDIA results, follow-up research was performed, extending the analysis to different sample types and to the different effects of long-term storage. The latter activity was in conjunction with the local European da Vinci Biobank. These results (which partially contributed to the ANNEX of CEN/TS 16945"MOLECULAR IN VITRO DIAGNOSTIC EXAMINATIONS - SPECIFICATIONS FOR PRE-EXAMINATION PROCESSES FOR METABOLOMICS IN URINE, VENOUS BLOOD SERUM AND PLASMA") are presented in detail
How toxic are gold nanoparticles? The state-of-the-art.
With the growing interest in biotechnological applications of gold nanoparticles and their effects exerted on the body, the possible toxicity is becoming an increasingly important issue. Numerous investigations carried out, in the last few years, under different experimental conditions, following different protocols, have produced in part conflicting results which have leaded to different views about the effective gold nanoparticle safety in human applications.
This work is intended to provide an overview on the most recent experimental results in order to summarize the current state-of-the-art. However, rather than to present a comprehensive review of the available literature in this field, that, among other things, is really huge, we have selected some representative examples of both in vivo and in vitro investigations, with the aim of offering a scenario from which clearly emerges the need of an urgent and impelling standardization of the experimental protocols. To date, despite the great potential, the safety of gold nanoparticles is highly controversial and important concerns have been raised with the need to be properly addressed. Factors such as shape, size, surface charge, surface coating and surface functionalization are expected to influence interactions with biological systems at different extents, with different outcomes, as far as gold nanoparticle potentiality in biomedical applications is concerned. Moreover, despite the continuous attempt to establish a correlation between structure and interactions with biological systems, we are still far from assessing the
toxicological profile of gold nanoparticles in an unquestionable manner. This review is intended to provide a contribution in this direction, offering some suggestions in order to reach the systematization of data over the most relevant physico-chemical parameters, which govern and control toxicity, at different cellular and organismal levels
Anticancer Effects of Red Raspberries on Immune Cells and Blood Parameters
There is an abundance of research correlating diets rich in fruits and vegetables to the reduction or prevention of chronic diseases such as cancer and cardiovascular disease. Plants extracts have also been claimed to possess antiviral, antibacterial, and immunological properties. This study focused on the in vitro and in vivo effects of a water extract from lyophilized Meeker red raspberries on tumor cell viability and immune parameters. A large number of studies have demonstrated the cytotoxic effect of different fruit juices and extracts against tumor cell lines in vitro. However, studies also show that the blood plasma levels of berry phytochemicals are several orders of magnitude lower than the levels which have activity in vitro. This study was undertaken as an attempt to reconcile these apparently contradictory observations and test the value of a novel system for detecting the effect of berry consumption in humans. Subjects who consumed berries donate blood which is tested in vitro. An extract of Meeker red raspberries was tested for its activity against 5 tumor cell lines. Berries from the same lot were consumed by test subjects and their plasma and white blood cells were tested for changes in several immune parameters. The extract exhibited a potent cytotoxic effect on a variety of cancer cells in vitro; including cells from gastric, prostate, colon, and breast cancers. By comparison to an ascorbic acid control, it could be determined that the cytotoxicity of the raspberry extract was not solely attributable to pH or antioxidant effects. The goal of the present study was to evaluate the effects of lyophilized red raspberry consumption on in vivo and in vitro immune parameters, including immune cell proliferation, plasma MMP-9 concentrations, and cytotoxicity toward human tumor cell lines. Sixteen healthy volunteers participated in the 3.5 day study. Venous blood samples were collected at baseline and after the last serving of berries. Although much variability was observed among participants, our results suggest that raspberry phytochemicals might augment immune function and affect both the innate and adaptive immune responses in some individuals, but have minimal effects in others. In several of the participants, the levels of one or more subsets of leukocytes changed after berry intake. In five donors mitogen-induced T lymphocyte proliferation increased and in five it decreased. An inverse relationship was observed between T lymphocyte mitogen stimulation and the change in leukocyte levels. There was an increase in resting peripheral blood mononuclear cell metabolism, suggesting an in vivo priming or proliferative effect from the berry phytochemicals. Changes in plasma levels of MMP-9 correlated with changes in leukocyte levels. In vitro plasma tumoricidal activity increased for all participants. An increase in peripheral blood mononuclear cell cytotoxicity was also observed in some donors. The results demonstrate that consumption of raspberry phytochemicals can affect immune parameters measured in vitro and may affect responses of the host to pathogenic challenge
Trapped ion mobility spectrometry and PASEF enable in-depth lipidomics from minimal sample amounts
A comprehensive characterization of the lipidome from limited starting material remains very challenging. Here we report a high-sensitivity lipidomics workflow based on nanoflow liquid chromatography and trapped ion mobility spectrometry (TIMS). Taking advantage of parallel accumulation-serial fragmentation (PASEF), we fragment on average 15 precursors in each of 100 ms TIMS scans, while maintaining the full mobility resolution of co-eluting isomers. The acquisition speed of over 100 Hz allows us to obtain MS/MS spectra of the vast majority of isotope patterns. Analyzing 1 mu L of human plasma, PASEF increases the number of identified lipids more than three times over standard TIMS-MS/MS, achieving attomole sensitivity. Building on high intra- and inter-laboratory precision and accuracy of TIMS collisional cross sections (CCS), we compile 1856 lipid CCS values from plasma, liver and cancer cells. Our study establishes PASEF in lipid analysis and paves the way for sensitive, ion mobility-enhanced lipidomics in four dimensions
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