Incorporation of SPION-casein core-shells into silk-fibroin nanofibers for cardiac tissue engineering

Mimicking the structure of extracellular matrix (ECM) of myocardium is necessary for fabrication of functional cardiac tissue. The superparamagnetic iron oxide nanoparticles (SPIONs, Fe₃O₄), as new generation of magnetic nanoparticles (NPs), are highly intended in biomedical studies. Here, SPION NPs (1 wt%) were synthesized and incorporated into silk-fibroin (SF) electrospun nanofibers to enhance mechanical properties and topography of the scaffolds. Then, the mouse embryonic cardiac cells (ECCs) were seeded on the scaffolds for in vitro studies. The SPION NPs were studied by scanning electron microscope (SEM), X-ray diffraction (XRD), and transmission electron microscope (TEM). SF nanofibers were characterized after incorporation of SPIONs by SEM, TEM, water contact angle measurement, and tensile test. Furthermore, cytocompatibility of scaffolds was confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SEM images showed that ECCs attached to the scaffolds with elongated morphologies. Also, the real-time PCR and immunostaining studies approved upregulation of cardiac functional genes in ECCs seeded on the SF/SPION-casein scaffolds including GATA-4, cardiac troponin T, Nkx 2.5, and alpha-myosin heavy chain, compared with the ones in SF. In conclusion, incorporation of core-shells in SF supports cardiac differentiation, while has no negative impact on ECCs' proliferation and self-renewal capacity.     

Circulating and tissue microRNAs as a potential diagnostic biomarker in patients with thrombotic events

Venous and arterial thrombosis are conditions that have a considerable burden if left untreated. The hypoxia-induced by the occluded vessel can disrupt the circulation of any organ, the cornerstone of treating thrombosis is rapid diagnosis and appropriate treatment. Diagnosis of thrombosis may be made by using laboratory tests or imaging techniques in individuals who have clinical manifestations of a thrombotic event. The use of serum micro ribonucleic acids (RNAs) has recently been applied to the diagnosis of thrombosis. These small RNA molecules are emerging as new diagnostic markers but have had very limited applications in vascular disease. Most of the articles provided various microRNAs with different levels of accuracy. However, there remains a lack of an appropriate panel of the most specific microRNA in the literature. The purpose of the present review was to summarize the existing data on the use of microRNAs as a diagnostic biomarker for venous thrombosis.     

Efficient growth inhibition of EGFR over-expressing tumor cells by an anti-EGFR nanobody

Epidermal growth factor receptor (EGFR) is deemed to be one of the main molecular targets for diagnosis and treatment of cancer. It has been identified that EGFR involves in pathogenesis of some forms of human cancers. Monoclonal antibodies targeting EGFR could control the tumor cell growth, proliferation, and apoptosis by suppressing the signal transduction pathways. Nanobodies can be regarded as the smallest intact antigen binding fragments, derived from heavy chain-only antibodies existing in camelids. Here, we describe the identification of an EGFR-specific nanobody, referred to as OA-cb6, obtained from immunized camel with a cell line expressing high levels of EGFR. Utilizing flow cytometry (FACS) and blotting methods, we demonstrated that OA-cb6 nanobody binds specifically to EGFR expressing on the surface of A431 cells. In addition, OA-cb6 nanobody potently causes the inhibition of EGFR over expression, cell growth and proliferation. The antibody fragments can probably be regarded as worthwhile binding block for further rational design of anti-cancer therapy.     

Probing the interaction of silver nanoparticles with tau protein and neuroblastoma cell line as nervous system models

Interestingly pharmaceutical sciences are using nanoparticles (NPs) to design and develop nanomaterials-based drugs. However, up to recently, it has not been well realized that NPs themselves may impose risks to the biological systems. In this study, the interaction of silver nanoparticles (AgNPs) with tau protein and SH-SY5Y neuroblastoma cell line, as potential nervous system models, was examined with a range of techniques including intrinsic fluorescence spectroscopy, circular dichroism (CD) spectroscopy, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and acridine orange/ethidium bromide (AO/EB) dual staining method. Fluorescence study showed that AgNPs with a diameter of around 10–20 nm spontaneously form a static complex with tau protein via hydrogen bonds and van der Waals interactions. CD experiment revealed that AgNPs did not change the random coil structure of tau protein. Moreover, AgNPs showed to induce SH-SY5Y neuroblastoma cell mortality through fragmentation of DNA which is a key feature of apoptosis. In conclusion, AgNPs may induce slight changes on the tau protein structure. Also, the concentration of AgNPs is the main factor which influences their cytotoxicity. Since, all adverse effects of NPs are not well detected, so probably additional more specific testing would be needed.     

Comparative immunomodulatory properties of adipose‐derived mesenchymal stem cells conditioned media from BALB/c,C57BL/6, and DBA mouse strains

Adipose tissue‐derived mesenchymal stem cells (AD‐MSCs) have been shown to be capable of differentiating into multiple cell type and exert immunomodulatory effects. Since the selection of ideal stem cell is apparently crucial for the outcome of experimental stem cell therapies, therefore, in this study we compared AD‐MSCs conditioned media (CM) from BALB/c, C57BL/6, and DBA mouse strains. No significant difference was found in the morphology, cell surface markers, in vitro differentiation and proliferation potentials of AD‐MSCs isolated from C57BL/6, BALB/c, and DBA mice. The immunological assays showed some variation among the strains in the cytokines, nitric oxide (NO), and indoleamine 2,3‐dioxygenase (IDO) production and immunomodulatory effects on splenocytes functions. Our results indicated a suppression of splenocytes proliferation in the presence of AD‐MSC CM from the three inbred mouse strains. However, BALB/c CM exerted a higher suppression of splenocytes proliferation. AD‐MSCs isolated from C57BL/6 and BALB/c mice produced higher levels of TGF‐β than those from DBA mice. Furthermore, IL‐17 and IDO production was higher in AD‐MSCs isolated from BALB/c mice. Our results indicated an increased production of TGF‐β, IL‐4, IL‐10, NO, and IDO by splenocytes in response to CM from BALB/c AD‐MSCs. In conclusion, our results showed that the immunomodulatory properties of mouse AD‐MSCs is strain‐dependent and this variation should be considered during selection of appropriate stem cell source for in vivo experiments and stem cell therapy strategies. J. Cell. Biochem. 114: 955–965, 2013. © 2012 Wiley Periodicals, Inc.     

Normalization of miRNA qPCR high-throughput data: a comparison of methods

Low-density quantitative real-time PCR (qPCR) arrays are often used to profile expression patterns of microRNAs in various biological milieus. To achieve accurate analysis of expression of miRNAs, non-biological sources of variation in data should be removed through precise normalization of data. We have systematically compared the performance of 19 normalization methods on different subsets of a real miRNA qPCR dataset that covers 40 human tissues. After robustly modeling the mean squared error (MSE) in normalized data, we demonstrate lower variability between replicates is achieved using various methods not applied to high-throughput miRNA qPCR data yet. Normalization methods that use splines or wavelets smoothing to estimate and remove Cq dependent non-linearity between pairs of samples best reduced the MSE of differences in Cq values of replicate samples. These methods also retained between-group variability in different subsets of the dataset.     

Revalidation of Phyllocaulis renschi (Mollusca: Gastropoda: Veronicellidae) based on radula and mandible structures of taxonomic significance for the genus

Exospermastix robustus Verhoeff, 1951 (Mato Grosso, nr Cuiaba) is regarded as a species of Urostreptus; the gonopods of the type specimen are illustrated and their structure discussed. The species is in many ways similar to U. camerani and U. paxillatus, but is much larger than either. The status of the generic and specific names Stenostreptus hassleri Carl is reviewed after a study of the type material, and the previous estimate of hassleri as a species of Urostreptus is reaffirmed. New drawings made from the lectotype of hassleri are given. The gonopods of a species identified as U. borelli by F. Silvestri, the author of that name, are illustrated, and the suggestion made that borelli may be found to be a senior synonym of hassleri.     

Motor Domain Phosphorylation Modulates Kinesin-1 Transport

Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated.     

Repetitive somatic embryogenesis from leaves of the medicinal plant Petiveria alliacea L.

Leaf segments from in vitro-grown shoot cultures of Petiveria alliacea were incubated on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin, thidiazuron, 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). Direct somatic embryogenesis was induced in response to all tested concentrations of 2,4-D and PIC. Primary somatic embryos displayed highly repetitive embryogenesis, both on the induction medium and in liquid hormone-free MS medium. Plantlets were obtained from these secondary embryos at an estimated frequency of 5 %, after 180 days of culture on half-strength MS medium gelled with 0.2 % Phytagel. Simultaneous development of friable non-embryogenic callus was also observed on media containing PIC or 2,4-D at different concentrations. Cell suspension cultures initiated from these callus tissues did not show an increase in biomass. The embryogenic portions formed at the surface of the explants in response to 20.0 μM PIC were inoculated in hormone-free full-or half-strength liquid MS medium (MS0) and showed high rates of secondary embryogenesis, resulting in the production of a mean of 35 embryos for each embryo inoculated at the culture initiation. Embryos that started the conversion process in the liquid MS0 medium originated whole plants at a frequency of 100 % when transferred to MS0 medium solidified with 0.7 % agar. Acclimatization was achieved in 90 % of the converted plantlets, with the production of phenotypically normal plants. This system is potentially useful for the micropropagation of this species, as well as for the production of substances with pharmacological interest, such as dibenzyl trisulfide.