Exosomes as a novel cell-free therapeutic approach in gastrointestinal diseases

Cell communication through extracellular vesicles (EVs) has been defined for many years and it is not limited only to neighboring cells, but also distant ones in organisms receive these signals. These vesicles are secreted from the variety of cells and are composed of a distinctive component such as proteins, lipids, and nucleic acids. EVs have different classified subgroups regarding their cell origin, in this context, exosomes are the most appealing particles in cell biology, especially clinical in recent years and are represented as novel therapeutic agents with numerous advantages alongside and/or over cell therapy. However, cell therapy had a hopeful outcome in gastrointestinal diseases which have minimal alternatives in their treatments. Inflammatory bowel disease (IBD), liver fibrosis, gastrointestinal cancers are the examples that cell therapy and immunotherapy were applied in their treatment, therefore, the cell products like exosomes are the beneficial option in their treatment even cancers with promising results in animal models. In this review, we consider the main defined biogenesis, function, and component of secreted exosomes in different cells with a specific focus on the potential application of these exosomes as a cell-free therapeutic approach in gastrointestinal diseases like IBD, gastric cancer, and colon cancer. Additionally, exosomes role as therapeutic reagents mainly mesenchymal stem cells and dendritic cell-derived exosomes in different studies have been under intense investigation and even they are being studied in different clinical trials. Therefore, all these striking functions described for secretome implies the importance of these biocarriers.     

Spike-timing in primary sensory neurons: a model of somatosensory transduction in the rat

In previous work, we constructed a simple electro-mechanical model of transduction in the rat mystacial follicle that was able to replicate primary afferent response profiles to a variety of whisker deflection stimuli. Here, we update that model to fit newly available spike-timing response data, and demonstrate that the new model produces appropriate responses to richer stimuli, including pseudo white noise and natural textures, at a spike-timing level of detail. Additionally, we demonstrate reliable distributed encoding of multi-component oscillatory signals. No modifications were necessary to the mechanical model of the physical components of the follicle–sinus complex, supporting its generality. We conclude that this model, and its continued development, will aid the understanding both of somatosensory systems in general, and of physiological results from higher (e.g. thalamocortical) systems by accurately characterising the signals on which they operate.     

The Mouse GalR2 Galanin Receptor: Genomic Organization, cDNA Cloning, and Functional Characterization

Abstract: The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity ( K _D = 0.47 n M ) and saturable binding with ¹²⁵I-galanin ( B _max = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin ⋍ M40 ⋍ M15 ⋍ M35 ⋍ C7 ⋍ galanin (2–29) ⋍ galanin (1–16) ≫ galanin (10–29) ⋍ galanin (3–29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.     

Citrinin detection by intensified fluorescence signal of a FRET-based immunosensor using magnetic/silica core–shell

The specific immune-reaction between the anti-citrinin antibody immobilized on the surface of magnetic/silica core–shell (MSCS) and the citrinin–Rho123–BSA conjugate brings the Rho123 fluorophore as an acceptor and the QDs as a donor in close spatial proximity and causes FRET for occurring upon photo-excitation of the QDs. The novelties of this study include: (1) immobilization of the MSCS; (2) large amount of the immobilized QDs, and (3) immobilization of a large amount of Rho123 on the BSA macromolecule. Cd/Te QDs were synthesized by the simultaneous reduction of cadmium chloride and tellurium in the presence of sodium borohydride. Magnetic nanoparticles were synthesized using FeSO₄ and FeCl₃. The prepared magnetic nanoparticles shelled by silica using tetraethoxysilane in the presence of ammonia. Transmission electron microscopy (TEM) analysis was used for investigating shape and monodispersity of the nanoparticles. EDC/NHS was used as a cross linking agent for immobilization of the QDs, conjugation of citrinin to amino groups of BSA, labeling of BSA with Rho123 and also for immobilization of the amino-functionalized MSCS on the immobilized QDs. Immobilization of the anti-citrinin antibody on the surface of the amino-functionalized MSCS was performed by Schiff-base mechanism. By using these three effective strategies, sensitivity of the designed nanobiosensor was incredibly enhanced as a very low limit of detection (up to 0.1 pM). The feasibility of this technique was tested by the detection of citrinin in the spiked human serum. Results showed that there was a linear correlation between the decreased fluorescence intensity of the Rho123 and increased fluorescence intensity of the QDs with increasing concentration of citrinin in the spiked samples in the range of 1–6 pM. According to obtained results, we conclude that this highly sensitive detection scheme is a easy, quick and impressive method that can be used in optical-based nanosensors.     

Genetic analysis of an F2 intercross between two strains of Japanese quail provided evidence for quantitative trait loci affecting carcass composition and internal organs

The purpose of this study was to identify genomic regions, quantitative trait loci (QTL), affecting carcass traits on chromosome 1 in an F2 population of Japanese quail. For this purpose, two white and wild strains of Japanese quail (16 birds) were crossed reciprocally and F1 generation (34 birds) was created. The F2 generation was produced by intercrossing of the F1 birds. Phenotypic data including carcass weight, internal organs and carcass parts were collected on F2 animals (422 birds). The total mapping population (472 birds) was genotyped for 8 microsatellite markers on chromosome 1. QTL analysis was performed with interval mapping method applying the line-cross model. Significant QTL were identified for breast weight at 0 (P < 0.01), 172 (P < 0.05) and 206 (P < 0.01), carcass weight at 91 (P < 0.05), carcass fatness at 0 (P < 0.01), pre-stomach weight at 206 (P < 0.01) and uropygial weight gland at 197 (P < 0.01) cM on chromosome 1. There was also evidence for imprinted QTL affecting breast weight (P < 0.01) on chromosome 1. The proportion of the F2 phenotypic variation explained by the significant additive, dominance and imprinted QTL effects ranged from 1.0 to 7.3 %, 1.2 to 3.3 % and 1.4 to 2.2 %, respectively.     

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.     

Evaluation of NUF2 and GMNN Expression in Prostate Cancer: Potential Biomarkers for Prostate Cancer Screening

Background:Prostate cancer (PC) is one of the most abundant cancers among men, and In Iran, has been responsible for 6% of all deaths from cancer in men. NUF2 and GMNN genes are considered as loci of susceptibility to tumorigenesis in humans. Alterations in expression of these genes have been reported in various malignancies. The aim of our study was to test whether different NUF2 and GMNN expression levels are associated with PC incidence and hence, might be considered as new molecular tools for PC screening.Methods:Biopsy samples from 40 PC patients and 41 healthy Iranian men were used to determine the relative gene expression. After RNA extraction and cDNA synthesis, samples were analyzed using TaqMan Quantitative Real time PCR. Patients’ background information, included smoking habits and family histories of PC, were recorded. Stages and grades of their PC were classified by the TNM tumor, node, metastasis (TMN) staging system based on standard guidelines.Results:NUF2 expression did not significantly differ between the groups, while GMNN expression was significantly greater in the PC specimens than in the controls.Conclusion:Regarding the significant role of GMNN in various tumor phenotypes, and its importance in PC progression, the alteration in GMNN expression in PC samples vs. controls indicate that the genetic profiling of this cancer might be considered to personalize therapy for each patient in the future.Key Words: Family history, Geminin (GMNN), Tumor staging, NUF2, Prostate cancer     

Kinetics of infected insect cell osmolysis and enhanced protein release using a modified disruption method

We have studied and characterized a cell disruption method to produce a protein extract from recombinant Baculovirus infected insect cells based on osmotic lysis. Cell lysis kinetics were measured during a 24-h incubation in lysis buffer and resulting data sets were curve fitted to a hyperbola, visually similar to the Michaelis–Menten curve, to determine the maximum concentration of released protein and the time required to reach equilibrium. Effect of parameters such as pH, ionic strength and infection phase were evaluated, and based on fittings optimal protein release conditions were obtained for total cell protein as well as the recombinant protein, HPV 16 L1. It was demonstrated that pH and the phase of infection can vastly influence the amount of release while ionic strength only effects the time required to achieve equilibrium in protein release. Osmolysis can be a mild, yet effective method to release recombinant protein with high recovery levels and hence can be used in capacities where stringent criteria regarding contamination with surfactant or non-cytoplasmic contents are observed.     

Tumor microenvironment: Interactions and therapy

Tumor microenvironment (TME) is a host for a complex network of heterogeneous stromal cells with overlapping or opposing functions depending on the dominant signals within this milieu. Reciprocal paracrine interactions between cancer cells with cells within the tumor stroma often reshape the TME in favor of the promotion of tumor. These complex interactions require more sophisticated approaches for cancer therapy, and, therefore, advancing knowledge about dominant drivers of cancer within the TME is critical for designing therapeutic schemes. This review will provide knowledge about TME architecture, multiple signaling, and cross communications between cells within this milieu, and its targeting for immunotherapy of cancer.