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In sustainable agriculture, there is a tendency for an increased use of microbiological preparations, especially plant growth promoting bacteria (PGPB), that can supplement the phenotypic plasticity and adaptability of plants, stimulate their growth and development, increase resistance to stress. The endophytic PGPB could be a promising element of technologies for the improvement of mineral nutrition and promotion of growth and yield of wheat (Triticum spp.). They are transferred to the plant by a horizontal, from the environment (rhizosphere, phyllosphere), or a vertical, from the seeds (from generation to generation), way. The growth-promoting effect of endophytes is mediated by the synthesis and secretion of phytohormones and secondary metabolites as well as their ability to absorb N2, suppress the development of bacterial and/or fungal phytopathogens; improve mineral nutrition. The review elucidates current data on the presence of bacterial endophytes in various organs of wheat plants and their characterization as potential PGPB. Data on the most common genera of bacterial endophytes of wheat (Bacillus, Micrococcus, Staphylococcus, Pseudomonas, Pantoea, Kosakonia, etc.) are presented, and their influence on plants is described, in particular, the effect on the absorption of micronutrients important for plants and humans such as iron (Fe) and zinc (Zn), resistance to stress factors and growth. The varietal differences in the wheat endophytic microbiome are noted. An increased micronutrient absorption and assimilation assisted by the bacterial endophytes are associated with the changes in endogenous auxins and ethylene, the release of organic acids, siderophores, indirect activation of metal transporters, etc. The mechanisms underlying plant growth stimulation are complex due to interactions between a microorganism and the whole plant microbiome and their changes during the plant ontogenesis. The analysis of the published data confirms the need for further studies of the species composition and mechanisms of interaction of endophytic PGPB to develop new strategies for improving mineral nutrition of wheat and trace element biofortification of grain. It is a feasible and promising technology of the future to overcome the problems of hidden hunger and provide quality food products to the world population with available resources and a reduced negative impact on the environment.

Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).

Spinal cord injury (SCI), is a devastating condition leading to the loss of locomotor and sensory function below the injured segment. Despite some progress in acute SCI treatment using stem cells and biomaterials, chronic SCI remains to be addressed. We have assessed the use of laminin-coated hydrogel with dual porosity, seeded with induced pluripotent stem cell-derived neural progenitors (iPSC-NPs), in a rat model of chronic SCI. iPSC-NPs cultured for 3 weeks in hydrogel in vitro were positive for nestin, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2). These cell-polymer constructs were implanted into a balloon compression lesion, 5 weeks after lesion induction. Animals were behaviorally tested, and spinal cord tissue was immunohistochemically analyzed 28 weeks after SCI. The implanted iPSC-NPs survived in the scaffold for the entire experimental period. Host axons, astrocytes and blood vessels grew into the implant and an increased sprouting of host TH+ fibers was observed in the lesion vicinity. The implantation of iPSC-NP-LHM cell-polymer construct into the chronic SCI led to the integration of material into the injured spinal cord, reduced cavitation and supported the iPSC-NPs survival, but did not result in a statistically significant improvement of locomotor recovery.

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1G93A transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1G93A rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs).

Due to high biocompatibility, miniaturization, optical transparency and low production cost together with high radiation hardness the diamond-based sensors are considered promising for radiation medicine and biomedicine in general. Here we present detection of fibroblast cell culture properties by nanocrystalline diamond solution-gated field-effect transistors (SG-FET), including effects of gamma irradiation. We show that blank nanocrystalline diamond field-effect biosensors are stable at least up to 300 Gy of γ irradiation. On the other hand, gate current of the diamond SG-FET biosensors with fibroblastic cells increases exponentially over an order of magnitude with increasing radiation dose. Extracellular matrix (ECM) formation is also detected and analyzed by correlation of electronic sensor data with optical, atomic force, fluorescence, and scanning electron microscopies.

Salicylic acid (SA) is considered one of the most important plant signaling molecules involved in abiotic and biotic stress tolerance. Application of 0,05 mM SA enhanced plants’ tolerance to cadmium stress by modulating levels of several metabolites, including components of antioxidative defense, osmolytes, secondary metabolites, and metal-chelating compounds. Seeds of bread wheat (Triticum aestivum L.) and buckwheat (Fagopyrum esculentum Moench) were presoaked in 0,05 mM SA/or distilled water for 5 h, with the following germination in a dark thermostat for 3 d. Uniform seedlings were planted into +Cd / or -Cd-containing substrate and grown for 16/8 h light/dark. It was revealed that seed pretreatment with SA alone caused a 3-5-fold decrease in phenylalanine ammonia-lyase (PAL) activity and total phenolic content compared to the control water treatment. Seed pretreatment with SA alleviated the negative impact of Cd on plant growth, PAL activity, and total phenolic content. These results indicate that salicylate treatment alleviates Cd toxicity by modifying lignin synthesis and accumulation of flavonoids in wheat and buckwheat plants. Protective effects were more pronounced for buckwheat with the studied variables' high total phenolic content.

Background Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder, characterized by a low platelet count ( Methods After a 2-week screening period, patients were randomized 1:1:1 to receive 4 weekly intravenous infusions of either placebo or efgartigimod at a dose of 5 mg/kg or 10 mg/kg. Following periods included 8-week follow-up, up to 13 weeks extended follow-up, and a 1-year open-label extension (OLE) where patients could be treated with efgartigimod 10 mg/kg (Figure 1). Patients aged 18 to 85 years with confirmed primary ITP, and an average of 2 platelet counts ˂30×109/L during screening (with no single reading >35×109/L), were recruited. Oral corticosteroids, oral immunosuppressants, and/or thrombopoietin receptor agonists at stable doses were permitted. The primary endpoint was safety. Secondary endpoints included pharmacodynamic (PD) markers, pharmacokinetic (PK) parameters, presence of anti-drug antibodies (ADA) and autoantibodies, and efficacy. Data for all endpoints were summarized by group using descriptive statistics. Data until the last visit of the first treatment cycle of the OLE period are reported. Results Thirty-eight patients (placebo [N=12], efgartigimod at a dose of 5 mg/kg [N=13] or 10 mg/kg [N=13]), mostly with longstanding ITP (median disease duration 4.8 [0.1-47.8] years) who had insufficient response to prior ITP therapy or failed splenectomy (N=6), were randomized. Twenty (52.6%) patients had a baseline platelet count Efgartigimod was well tolerated with no dose-related safety observations and the safety profile was consistent with previous observations in healthy volunteers. No increased risk of infection was apparent. Low positive pre- and post-dose ADA titers were measured in all groups and did not have an apparent effect on PK/PD parameters. Treatment with efgartigimod resulted in a rapid reduction of total IgG (up to 63.7% mean change from baseline) and all IgG subtypes in all treated patients. Autoantibodies were identified in all patients and were reduced following efgartigimod treatment. Efgartigimod-treated groups achieved a higher maximum mean platelet count change from baseline compared to the placebo group. A higher proportion of efgartigimod-treated patients achieved a platelet count of ≥50×109/L and ≥100×109/L at any time compared to the placebo group (Figure 2). Forty-six percent patients on efgartigimod vs. 25% on placebo achieved a platelet count of ≥50×109/L on at least 2 occasions and 38% vs. 0% achieved ≥50×109/L for at least 10 cumulative days. A decreased incidence of bleeding, measured using WHO and ITP-BAT scales, was observed in both efgartigimod-treated groups. In the OLE period, in which 12 patients received efgartigimod 10 mg/kg, 8 (66.7%) patients achieved platelet count ≥50×109/L on at least 2 occasions. Conclusion A short 3-week treatment cycle of efgartigimod in patients with ITP predominantly refractory to previous lines of ITP therapy was well tolerated, markedly reduced IgG levels, was associated with clinically relevant increases in platelet counts, and reduced the proportion of patients with bleeding (Figure 3). This suggests that targeted IgG reduction with efgartigimod is a potential new treatment modality in primary ITP, and warrants evaluation of longer-term treatment in a larger Phase 3 study. Disclosures Newland: Novartis: Consultancy, Honoraria, Research Funding; Angle: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Ono: Consultancy, Honoraria; Shionogi: Consultancy, Honoraria; Rigel: Consultancy, Honoraria, Research Funding; Argenx: Consultancy, Honoraria; Dova: Consultancy, Honoraria. Sánchez-González:Amgen: Consultancy, Speakers Bureau; Gilead: Speakers Bureau; Navartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Godar:Argenx: Employment. Verschueren:Argenx: Consultancy. Gandini:Argenx: Employment, Equity Ownership. Ulrichts:Argenx: Employment. Beauchamp:Argenx: Employment. Dreier:Argenx: Employment. Ward:Argenx: Equity Ownership, Honoraria, Research Funding. Michel:Rigel: Consultancy; Amgen: Consultancy; Novartis: Consultancy. Liebman:Pfizer: Consultancy; Dova: Consultancy; Bristol-Myers: Research Funding; Argenx: Consultancy; Rigel: Consultancy; Janssen: Research Funding; Novartis: Consultancy. De Haard:Argenx: Employment, Equity Ownership, Patents & Royalties. Leupin:Argenx: Employment, Equity Ownership, Patents & Royalties. Kuter:Dova: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; UCB: Consultancy, Honoraria; Momenta: Consultancy, Honoraria; Caremark: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Protalix: Consultancy, Honoraria; Principia: Consultancy, Honoraria, Research Funding; Platelet Disorder Support Association: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Principia: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Kyowa-Kirin: Consultancy, Honoraria; Kyowa-Kirin: Consultancy, Honoraria; Merck Sharp Dohme: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Merck Sharp Dohme: Consultancy, Honoraria; Protalix: Consultancy, Honoraria; Rigel: Consultancy, Honoraria, Research Funding; Rigel: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Bristol Myers Squibb (BMS): Consultancy, Honoraria, Research Funding; Alnylam: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Protalex: Consultancy, Honoraria, Research Funding; Dova: Consultancy, Honoraria; Protalex: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Up-to-Date: Consultancy, Honoraria, Patents & Royalties: 3 Up-to-Date chapters; Up-to-Date: Consultancy, Honoraria, Patents & Royalties: 3 Up-to-Date chapters; Zafgen: Consultancy, Honoraria; Takeda (Bioverativ): Consultancy, Honoraria, Research Funding; Platelet Disorder Support Association: Consultancy, Honoraria; Shinogi: Consultancy, Honoraria; Agios: Consultancy, Honoraria, Research Funding; Actelion (Syntimmune): Consultancy, Honoraria, Research Funding; Takeda (Bioverativ): Consultancy, Honoraria, Research Funding; Caremark: Consultancy, Honoraria; Kezar: Research Funding; Kezar: Research Funding; Shire: Consultancy, Honoraria; Argenx: Consultancy, Honoraria, Research Funding; Alnylam: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb (BMS): Consultancy, Honoraria, Research Funding; Argenx: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Actelion (Syntimmune): Consultancy, Honoraria, Research Funding; Shinogi: Consultancy, Honoraria; Zafgen: Consultancy, Honoraria; Momenta: Consultancy, Honoraria; UCB: Consultancy, Honoraria.

Human embryonic stem cell-derived neural precursors (hESC NPs) are considered to be a promising tool for cell-based therapy in central nervous system injuries and neurodegenerative diseases. The Ca(2+) ion is an important intracellular messenger essential for the regulation of various cellular functions. We investigated the role and physiology of Ca(2+) signaling to characterize the functional properties of CCTL14 hESC NPs during long-term maintenance in culture (in vitro). We analyzed changes in cytoplasmic Ca(2+) concentration ([Ca(2+)]i) evoked by high K(+), adenosine-5'-triphosphate (ATP), glutamate, γ-aminobutyric acid (GABA), and caffeine in correlation with the expression of various neuronal markers in different passages (P6 through P10) during the course of hESC differentiation. We found that only differentiated NPs from P7 exhibited significant and specific [Ca(2+)]i responses to various stimuli. About 31% of neuronal-like P7 NPs exhibited spontaneous [Ca(2+)]i oscillations. Pharmacological and immunocytochemical assays revealed that P7 NPs express L- and P/Q-type Ca(2+) channels, P2X2, P2X3, P2X7, and P2Y purinoreceptors, glutamate receptors, and ryanodine (RyR1 and RyR3) receptors. The ATP- and glutamate-induced [Ca(2+)]i responses were concentration-dependent. Higher glutamate concentrations (over 100 μM) caused cell death. Responses to ATP were observed in the presence or in the absence of extracellular Ca(2+). These results emphasize the notion that with time in culture, these cells attain a transient period of operative Ca(2+) signaling that is predictive of their ability to act as stem elements.

Introduction A growing number of studies have highlighted the potential of stem cell and more-differentiated neural cell transplantation as intriguing therapeutic approaches for neural repair after spinal cord injury (SCI). Methods A conditionally immortalized neural stem cell line derived from human fetal spinal cord tissue (SPC-01) was used to treat a balloon-induced SCI. SPC-01 cells were implanted into the lesion 1 week after SCI. To determine the feasibility of tracking transplanted stem cells, a portion of the SPC-01 cells was labeled with poly-L-lysine-coated superparamagnetic iron-oxide nanoparticles, and the animals grafted with labeled cells underwent magnetic resonance imaging. Functional recovery was evaluated by using the BBB and plantar tests, and lesion morphology, endogenous axonal sprouting and graft survival, and differentiation were analyzed. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted SPC-01 cells on endogenous regenerative processes. Results Transplanted animals displayed significant motor and sensory improvement 2 months after SCI, when the cells robustly survived in the lesion and partially filled the lesion cavity. qPCR revealed the increased expression of rat and human neurotrophin and motor neuron genes. The grafted cells were immunohistologically positive for glial fibrillary acidic protein (GFAP); however, we found 25% of the cells to be positive for Nkx6.1, an early motor neuron marker. Spared white matter and the robust sprouting of growth-associated protein 43 (GAP43)+ axons were found in the host tissue. Four months after SCI, the grafted cells matured into Islet2+ and choline acetyltransferase (ChAT)+ neurons, and the graft was grown through with endogenous neurons. Grafted cells labeled with poly-L-lysine-coated superparamagnetic nanoparticles before transplantation were detected in the lesion on T2-weighted images as hypointense spots that correlated with histologic staining for iron and the human mitochondrial marker MTCO2. Conclusions The transplantation of SPC-01 cells produced significant early functional improvement after SCI, suggesting an early neurotrophic action associated with long-term restoration of the host tissue, making the cells a promising candidate for future cell therapy in patients with SCI.

Introduction The use of immortalized neural stem cells either as models of neural development in vitro or as cellular therapies in central nervous system (CNS) disorders has been controversial. This controversy has centered on the capacity of immortalized cells to retain characteristic features of the progenitor cells resident in the tissue of origin from which they were derived, and the potential for tumorogenicity as a result of immortalization. Here, we report the generation of conditionally immortalized neural stem cell lines from human fetal spinal cord tissue, which addresses these issues. Methods Clonal neural stem cell lines were derived from 10-week-old human fetal spinal cord and conditionally immortalized with an inducible form of cMyc. The derived lines were karyotyped, transcriptionally profiled by microarray, and assessed against a panel of spinal cord progenitor markers with immunocytochemistry. In addition, the lines were differentiated and assessed for the presence of neuronal fate markers and functional calcium channels. Finally, a clonal line expressing eGFP was grafted into lesioned rat spinal cord and assessed for survival, differentiation characteristics, and tumorogenicity. Results We demonstrate that these clonal lines (a) retain a clear transcriptional signature of ventral spinal cord progenitors and a normal karyotype after extensive propagation in vitro, (b) differentiate into relevant ventral neuronal subtypes with functional T-, L-, N-, and P/Q-type Ca2+ channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity. Conclusions We propose that these cells represent a useful tool both for the in vitro study of differentiation into ventral spinal cord neuronal subtypes, and for examining the potential of conditionally immortalized neural stem cells to facilitate functional recovery after spinal cord injury or disease.

Currently, there is no effective strategy for the treatment of spinal cord injury (SCI). A combination of biomaterials and stem cell therapy seems to be a promising approach to increase regenerative potential after SCI. We evaluated the use of a cellpolymer construct based on a combination of the conditionally immortalized spinal progenitor cell line SPC-01_GFP3, derived from human fetal spinal cord tissue, with a serotonin-modified poly(2-hydroxyethyl methacrylate) hydrogel (pHEMA-5HT). We compared the effect of treatment with a pHEMA-5HT hydrogel seeded with SPC-01_GFP3 cells, treatment with a pHEMA-5HT only and no treatment on functional outcome and tissue reconstruction in hemisected rats. Prior to transplantation the cell-polymer construct displayed a high potential to support the growth, proliferation and differentiation of SPC-01 cells in vitro. One month after surgery, combined hydrogel-cell treatment reduced astrogliosis and tissue atrophy and increased axonal and blood vessel ingrowth into the implant; however, two months later only the ingrowth of blood vessels remained increased. SPC-01_GFP3 cells survived well in vivo and expressed advanced markers of neuronal differentiation. However, a majority of the transplanted cells migrated out of the lesion and only rarely remained in the hydrogel. No differences among the groups in motor or sensory recovery were observed. Despite the support of the hydrogel as a cell carrier in vitro, and good results in vivo one month postsurgery, there was only a small effect on long term recovery, mainly due to the limited ability of the hydrogels to support the in vivo growth and differentiation of cells within the implant. Further modifications will be necessary to achieve stable long term improvement in functional outcome.

Background Stem cell treatment provides a promising therapy for patients with spinal cord injury (SCI). However, the applied stem cells exert their effects in different manners that are dependent on the route used for administration. Methods In the present study, we administered neural precursors derived from induced pluripotent stem cells (iPS-NPs) either intraspinally into the lesion center or intrathecally into the subarachnoid space of rats with a balloon-induced spinal cord compression lesion. Functional locomotor performance, cell survival, astrogliosis, axonal sprouting and the expression of endogenous neurotrophic growth factors were evaluated using behavioral tests (BBB, flat beam test, rotarod, plantar test), morphometric analysis, immunohistochemistry and qPCR. Results Both treatments facilitated the functional locomotor recovery of rats with SCI. iPS-NPs injected intraspinally survived well for 2 months and were positive for MAP2, while cells grafted intrathecally were undetectable at the site of administration or in the spinal cord tissue. Intraspinal implantation increased gray and white matter sparing and axonal sprouting and reduced astrogliosis, while intrathecal application resulted only in an improvement of white matter sparing and an increase in axonal sprouting, in parallel with no positive effect on the expression of endogenous neurotrophic growth factor genes or glial scar reduction. Conclusions Intrathecally grafted iPS-NPs had a moderate therapeutic benefit on SCI through a paracrine mechanism that does not require the cells to be present in the tissue; however, the extended survival of i.s. grafted cells in the spinal cord may promote long-term spinal cord tissue regeneration.