New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n?=?16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n?=?16) after operation were cooled to 5 °C for 24 h, then frozen after 24 h pre-cooling to 5 °C, thawed and just after thawing their quality was analyzed. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.

     

Molecular Dynamics Study of Escherichia coli Thymidine Phosphorylase in a Complex with 3'-Azidothymidine Inhibitor and Phosphate

Russian Journal of Bioorganic Chemistry - Using a molecular dynamics method, the state of the dimeric thymidine phosphorylase molecule from Escherichia coli in a complex with noncompetitive enzyme...     

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates

BACKGROUNDThe development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI.AIMTo investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs).METHODSSeven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group (n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control (n = 3) was injected identically with the equivalent volume of vehicle.RESULTSFollow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones.CONCLUSIONOur data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes.     

Oxidation-induced calcium-dependent dehydration of normal human red blood cells

Phenazine-methosulphate (PMS) is a strong oxidant that induces reactive oxygen species (ROS) formation in cells. Though it has been shown that PMS increases the red blood cell (RBC) membrane permeability to K⁺, the hypotheses on the mechanism of PMS-induced effects are contradictory and there are no data on volume changes induced by this oxidant. Therefore, the influence of the PMS + ascorbate oxidative system on the volume of normal human RBCs was studied. In a Ca^{2 +} -containing medium, PMS + ascorbate caused dehydration (shrinking) of RBCs judged by: (1) changes in the density and osmotic resistance distributions of RBCs, and (2) a decrease in their low-angle scattering assessed by FACS analysis. The dehydration resulted from activation of the Gardos channels, was PMS and ascorbate concentration-dependent, was associated with broadening of the density and osmotic resistance distributions of the RBCs, and decreased in the presence of the taxifolin and rutin antioxidants. These findings contribute to a better understanding of the physiology and pathology of oxidatively-modified RBCs and may be of practical significance in estimating the antioxidant activity of various substances.     

Peptide Mimetic of the S100A4 Protein Modulates Peripheral Nerve Regeneration and Attenuates the Progression of Neuropathy in Myelin Protein P0 Null Mice

We recently found that S100A4, a member of the multifunctional S100 protein family, protects neurons in the injured brain and identified two sequence motifs in S100A4 mediating its neurotrophic effect. Synthetic peptides encompassing these motifs stimulated neuritogenesis and survival in vitro and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration and survival of myelinated axons. H3 accelerated electrophysiological, behavioral and morphological recovery after sciatic nerve crush while transiently delaying regeneration after sciatic nerve transection and repair. On the basis of the finding that both S100A4 and H3 increased neurite branching in vitro, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P₀ null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1 disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss, such as Charcot-Marie-Tooth type 1 disease. Moreover, our data suggest that S100A4 is a neuroprotectant in PNS and that other S100 proteins, sharing high homology in the H3 motif, may have important functions in PNS pathologies.     

Synthesis and DNA Duplex Stabilities of Oligonucleotides Containing C-5-(3-Methoxypropynyl)-2′-deoxyuridine Residues

Abstract

5′-Dimethoxytrityl-5-(3-methoxypropynyl)-2′-deoxyuridine phosphoroamidite was synthesized with the use of commercial 3- methoxypropyne. Oligonucleotides (ODNs) containing 5-(3- ethoxypropynyl)- 2′-deoxyuridine in different positions were prepared. The stabilities of the duplexes formed by these ODNs with the complementary templates are increased in comparison with the unmodified counterparts. On average modified residue incorporated, the Tm is raised by 1°C.     

Reagents For The Selective Immobilization Of Oligonucleotides On Solid Supports

New reagents (CPGs and phosphoramidites) for automatic solid phase synthesis of modified oligonucleotides were designed. Three oligonucleotides carrying fluorescent label at the 5′-terminus and an anchor group at the 3′-terminus were prepared and their immobilization in orthogonal conditions on solid supports was studied.     

Synthesis and Application of Negatively Charged PNA Analogues

Negatively charged DNA mimics containing phosphonate analogues of peptide nucleic acids were designed, and their physicochemical and biological properties were evaluated in the comparison with natural oligonucleotides, classical peptide nucleic acids, and morpholino phosphorodiamidate oligonucleotide analogues. The results obtained revealed a high potential of phosphonate-containing PNA derivatives for a number of biological applications, such as diagnostic, nucleic acids analysis, and inhibition of gene expression.