Distribution of biodiversity of wild beet species (Genus Beta L.) in Armenia under ongoing climate change conditions

The reported annual temperature increase and significant precipitation drop in Armenia impact the country’s ecosystems and biodiversity. The present study surveyed the geographical distribution of the local wild beet species under the ongoing climate change conditions. We showed that B. lomatogona, B. corolliflora and B. macrorhiza are sensitive to climate change and were affected to various degrees, depending on their location. The most affected species was B. lomatogona, which is at the verge of extinction. Migration for ca. 90 and 200–300 m up the mountain belt was recorded for B. lomatogona and B. macrorhiza, respectively. B. corolliflora was found at 100–150 m lower altitudes than in the 1980s. A general reduction in the beet’s population size in the native habitats was observed, with an increased number of plants within the populations, recorded for B. corolliflora and B. macrorhiza. A new natural hybrid Beta x intermedium Aloyan between B. corolliflora and B. macrorhiza was described and confirmed using chloroplast DNA trnL-trnF intergenic spacer (LF) and partially sequenced alcohol dehydrogenase (adh) of nuclear DNA. An overview of the wild beets reported in Armenia with the taxonomic background, morphological features, and distribution is provided. Conservation measures for preservation of these genetic resources are presented.publishedVersio

Iron Labeling and Pre-Clinical MRI Visualization of Therapeutic Human Neural Stem Cells in a Murine Glioma Model

Treatment strategies for the highly invasive brain tumor, glioblastoma multiforme, require that cells which have invaded into the surrounding brain be specifically targeted. The inherent tumor-tropism of neural stem cells (NSCs) to primary and invasive tumor foci can be exploited to deliver therapeutics to invasive brain tumor cells in humans. Use of the strategy of converting prodrug to drug via therapeutic transgenes delivered by immortalized therapeutic NSC lines have shown efficacy in animal models. Thus therapeutic NSCs are being proposed for use in human brain tumor clinical trials. In the context of NSC-based therapies, MRI can be used both to non-invasively follow dynamic spatio-temporal patterns of the NSC tumor targeting allowing for the optimization of treatment strategies and to assess efficacy of the therapy. Iron-labeling of cells allows their presence to be visualized and tracked by MRI. Thus we aimed to iron-label therapeutic NSCs without affecting their cellular physiology using a method likely to gain United States Federal Drug Administration (FDA) approval.For human use, the characteristics of therapeutic Neural Stem Cells must be clearly defined with any pertubation to the cell including iron labeling requiring reanalysis of cellular physiology. Here, we studied the effect of iron-loading of the therapeutic NSCs, with ferumoxide-protamine sulfate complex (FE-Pro) on viability, proliferation, migratory properties and transgene expression, when compared to non-labeled cells. FE-Pro labeled NSCs were imaged by MRI at tumor sites, after intracranial administration into the hemisphere contralateral to the tumor, in an orthotopic human glioma xenograft mouse model.FE-Pro labeled NSCs retain their proliferative status, tumor tropism, and maintain stem cell character, while allowing in vivo cellular MRI tracking at 7 Tesla, to monitor their real-time migration and distribution at brain tumor sites. Of significance, this work directly supports the use of FE-Pro-labeled NSCs for real-time tracking in the clinical trial under development: "A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically modified Neural Stem Cells Expressing Escherichia coli Cytosine Deaminase for Treatment of Recurrent High-Grade Gliomas"

The Involvement of Insulin-Like Growth Factor 1 and Nerve Growth Factor in Alzheimer’s Disease-Like Pathology and Survival Role of the Mix of Embryonic Proteoglycans: Electrophysiological Fingerprint, Structural Changes and Regulatory Effects on Neurotrophins

Alzheimer’s disease (AD)-associated neurodegeneration is triggered by different fragments of amyloid beta (Aβ). Among them, Aβ (25–35) fragment plays a critical role in the development of neurodegeneration—it reduces synaptic integrity by disruption of excitatory/inhibitory ratio across networks and alters the growth factors synthesis. Thus, in this study, we aimed to identify the involvement of neurotrophic factors—the insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF)—of AD-like neurodegeneration induced by Aβ (25–35). Taking into account our previous findings on the neuroprotective effects of the mix of proteoglycans of embryonic genesis (PEG), it was suggested to test its regulatory effect on IGF-1 and NGF levels. To evaluate the progress of neurodegeneration, in vivo electrophysiological investigation of synaptic activity disruption of the entorhinal cortex–hippocampus circuit at AD was performed and the potential recovery effects of PEG with relative structural changes were provided. To reveal the direct effects of PEG on brain functional activity, the electrophysiological pattern of the single cells from nucleus supraopticus, sensomotor cortex and hippocampus after acute injection of PEG was examined. Our results demonstrated that after i.c.v. injection of Aβ (25–35), the level of NGF decreased in cerebral cortex and hypothalamus, and, in contrast, increased in hippocampus, prompting its multidirectional role in case of brain damage. The concentration of IGF-1 significantly increased in all investigated brain structures. The administration of PEG balanced the growth factor levels accompanied by substantial restoration of neural tissue architecture and synaptic activity. Acute injection of PEG activated the hypothalamic nucleus supraopticus and hippocampal neurons. IGF-1 and NGF levels were found to be elevated in animals receiving PEG in an absence of amyloid exposure. We suggest that IGF-1 and NGF play a critical role in the development of AD. At the same time, it becomes clear that the neuroprotective effects of PEG are likely mediated via the regulation of neurotrophins

Sensitivity of MRI monitoring of FE-Pro-labeled NSCs targeting human glioma.

<p>(A) T2-weighted MR image of mouse brain in Fomblin, showing two distinct signal voids generated by FE-Pro-labeled NSCs that were injected ∼200 µm apart from each other on the left hemisphere and a hypointense signal generated by FE-Pro-labeled NSCs that migrated to the contralateral tumor site (white dotted boxes). Approximately 600 FE-Pro-labeled NSCs constituted a detectable signal void. (B and C) Prussian blue stained section from the region shown in (A). Higher magnification images (B, tumor area denoted by black dotted line) of the regions outlined in (C), showing PB-positive labeled NSCs corresponding to the hypointense signal sites in (A). MRI conditions: 7.0 Tesla, Rapid Acquisition Relaxation Enhancement sequence, 78 µm/pixel, 300 µm/slice, T_R/T_E = 1500/23.1 ms. Scale bars = 200 µm (B), 500 µm (C).</p

Retention of FE-Pro label in HB1.F3.CD NSCs.

<p>Data is displayed as means +/− SD of Prussian blue positive iron-loaded NSCs (% of total cell number). The data were obtained from 5 random fields of each independently labeled triplicate sample at 24, 48 and 96 h post-labeling.</p

Cellular viability of FE-Pro-labeled NSCs.

<p>(A) Cellular biomass normalized to non-labeled NSC cell growth at day 1 as measured by absorbance of protein-bound sulforhodamine B (SRB) at 570 nm. Data are mean±SE of triplicate samples and were analyzed using paired t-test between non-labeled vs. each FE-Pro dosage. P<0.05 was considered statistically significant. (B) Representative FACS plots showing the viable and apoptotic cell populations at 24 hours post-label and before sub-culturing. (C–D) Bar graphs showing the percentage of healthy cells at days 1, 4 and 8 for non-labeled NSCs (C), and FE-Pro-labeled NSCs (D) after sub-culturing passage at each time point. (E): Confocal images of healthy FE-Pro labeled and non-labeled NSCs (left panel) and apoptosis-induced FE-Pro labeled and non-labeled NSCs (right panel) at Day 6 post-labeling. Staining: PI (red), YO-Pro-1 (green). A FE-Pro dosage of 50∶3 µg/ml was used for each labeled sample unless otherwise indicated. Abbreviations: FE-Pro, Ferumoxide-Protamine Sulfate complex; PI, propidium iodide; Magnification: 20×.</p

Labeling efficiency of FE-Pro.

<p>(A) Light microscopy images of Prussian blue-stained non-labeled and FE-Pro-labeled NSCs at 24, 48 and 96 hours after labeling. (B) Electron micrographs of Fe-Pro-labeled NSCs. (C) Higher magnification image of outlined area in (B). Red arrows point to internalized FE-Pro complex in membrane-bound organelles. (D–E) T2-weighted MR images of labeled (L), non-labeled (N), and an equal mixture (M) of NSCs grown in soft agar. Each phantom contained three different total numbers of NSCs (1×10⁴, 1×10⁵ and 5×10⁵) each in 500 µl of 20% DMEM and 0.8% agar. Coronal view (D) and axial view at 5×10⁵ (E. left) and 1×10⁵ (E. right) of the phantoms. Decrease in T2-w signal strength correlated with the number of labeled cells in the phantom. (F) Graph of T2-w signal intensity vs. number of labeled NSCs. Data were extracted from 5 random fields of each corresponding phantom using ImageJ and shown as mean±SE. MRI conditions: 7.0 Tesla, Gradient-Echo sequence, voxel size = 0.09 mm³, TR/TE = 5402.5/90 ms. Scale bars = 50 µm (A), 2 µm (B) and 200 nm (C).</p

Functionality of FE-Pro labeled NSCs.

<p>(A) Results from Boyden chamber migration assays, showing inherent NSC migration towards conditioned media from U251 (media collected at 24 and 48 hours), UPN029, U87, and U87ffluc cell lines. P<0.05 was considered statistically significant. (B) Flow cytometry plot, showing expression of Cytosine Deaminase (CD) in non-labeled (red (isotype control) and green (anti-bCD)) and FE-Pro-labeled (brown (isotype control) and blue (anti-b-CD)) HB1.F3.CD cells. Abbreviations: HB1.F3.CD.FE-Pro, FE-Pro-labeled HB1.F3.CD NSCs; Anti-bCD, anti-bacterial CD primary antibody.</p