Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources

Background aimsMesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency.MethodsThe authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics.ResultsMSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications.ConclusionsThe authors’ data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products.     

Lactate-Modulated Induction of THBS-1 Activates Transforming Growth Factor (TGF)-beta2 and Migration of Glioma Cells In Vitro

Background

An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein.

Methods

Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays.

Results

Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells.

Conclusion

We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.     

Automated Detection Framework of the Calcified Plaque with Acoustic Shadowing in IVUS Images

Intravascular Ultrasound (IVUS) is one ultrasonic imaging technology to acquire vascular cross-sectional images for the visualization of the inner vessel structure. This technique has been widely used for the diagnosis and treatment of coronary artery diseases. The detection of the calcified plaque with acoustic shadowing in IVUS images plays a vital role in the quantitative analysis of atheromatous plaques. The conventional method of the calcium detection is manual drawing by the doctors. However, it is very time-consuming, and with high inter-observer and intra-observer variability between different doctors. Therefore, the computer-aided detection of the calcified plaque is highly desired. In this paper, an automated method is proposed to detect the calcified plaque with acoustic shadowing in IVUS images by the Rayleigh mixture model, the Markov random field, the graph searching method and the prior knowledge about the calcified plaque. The performance of our method was evaluated over 996 in-vivo IVUS images acquired from eight patients, and the detected calcified plaques are compared with manually detected calcified plaques by one cardiology doctor. The experimental results are quantitatively analyzed separately by three evaluation methods, the test of the sensitivity and specificity, the linear regression and the Bland-Altman analysis. The first method is used to evaluate the ability to distinguish between IVUS images with and without the calcified plaque, and the latter two methods can respectively measure the correlation and the agreement between our results and manual drawing results for locating the calcified plaque in the IVUS image. High sensitivity (94.68%) and specificity (95.82%), good correlation and agreement (>96.82% results fall within the 95% confidence interval in the Student t-test) demonstrate the effectiveness of the proposed method in the detection of the calcified plaque with acoustic shadowing in IVUS images.     

An infrared sensor analysing label‐free the secondary structure of the Abeta peptide in presence of complex fluids

The secondary structure change of the Abeta peptide to beta‐sheet was proposed as an early event in Alzheimer's disease. The transition may be used for diagnostics of this disease in an early state. We present an Attenuated Total Reflection (ATR) sensor modified with a specific antibody to extract minute amounts of Abeta peptide out of a complex fluid. Thereby, the Abeta peptide secondary structure was determined in its physiological aqueous environment by FTIR‐difference‐spectroscopy. The presented results open the door for label‐free Alzheimer diagnostics in cerebrospinal fluid or blood. It can be extended to further neurodegenerative diseases.

An immunologic ATR‐FTIR sensor for Abeta peptide secondary structure analysis in complex fluids is presented.     

Temporal Dynamics of Powdery Mildew (Oidium neolycopersici) and its Effects on the Host Growth Dynamics of Tomato

Controlled glasshouse experiments were conducted to investigate the temporal progress of powdery mildew and its effects on host dynamics of tomato, without and with one fungicide application. Healthy tomato transplants (5‐ to 6‐week old) were artificially inoculated with powdery mildew, and disease progress as well as host growth were monitored in both fungicide sprayed and unsprayed treatments and compared with non‐inoculated plants. Actual disease severity on a plant basis increased in unsprayed plants reaching maximum severity in the proportionate range of 0.53–0.83. One fungicide spray significantly reduced the maximum disease severity by two‐ to fourfolds. Despite adjustments for defoliation, declines in the proportion of disease severity between successive assessments were evident. Whereas the estimated growth rates of diseased plants were significantly lower than that of healthy plants, no significant differences were observed in the maximum leaf area formed of inoculated and non‐inoculated plants. A considerable effect of the powdery mildew epidemics was manifested through hastened shrivelling and defoliation of diseased leaves within the tomato canopy. An average of 18–29% and 40–52% of leaves had abscised from the plant canopy at the last date of assessment in sprayed and non‐sprayed plants, respectively. Accordingly, defoliation accounted for 14–33.3% and 58.3–63.1% losses in leaf area of sprayed and non‐sprayed plants, respectively. Duration of healthy leaf area and yield of inoculated plants were also significantly reduced by powdery mildew epidemics.     

Systematic research on the pretreatment of peptides for quantitative proteomics using a C18 microcolumn

Reversed phase microcolumns have been widely used for peptide pretreatment to desalt and remove interferences before tandem LC–MS in proteomics studies. However, few studies have characterized the effects of experimental parameters as well as column characteristics on the composition of identified peptides. In this study, several parameters including the concentration of ACN in washing buffer, the microcolumn's purification effect, the peptide recovery rate, and the dynamic‐binding capacity were characterized in detail, based upon stable isotope labeling by amino acids in a cell culture quantitative approach. The results showed that peptide losses can be reduced with low ACN concentration in washing buffers resulting in a recovery rate of approximately 82%. Furthermore, the effects of ACN concentration and loading amount on the properties of identified peptides were also evaluated. We found that the dynamic‐binding capacity of the column was approximately 26 μg. With increased loading amounts, more hydrophilic peptides were replaced by hydrophobic peptides.     

Thin‐Film Solar Cells with InP Absorber Layers Directly Grown on Nonepitaxial Metal Substrates

The design and performance of solar cells based on InP grown by the nonepitaxial thin‐film vapor–liquid–solid (TF‐VLS) growth technique is investigated. The cell structure consists of a Mo back contact, p‐InP absorber layer, n‐TiO₂ electron selective contact, and indium tin oxide transparent top electrode. An ex situ p‐doping process for TF‐VLS grown InP is introduced. Properties of the cells such as optoelectronic uniformity and electrical behavior of grain boundaries are examined. The power conversion efficiency of first generation cells reaches 12.1% under simulated 1 sun illumination with open‐circuit voltage (V_OC) of 692 mV, short‐circuit current (J_SC) of 26.9 mA cm^?2, and fill factor (FF) of 65%. The FF of the cell is limited by the series resistances in the device, including the top contact, which can be mitigated in the future through device optimization. The highest measured V_OC under 1 sun is 692 mV, which approaches the optically implied V_OC of ≈795 mV extracted from the luminescence yield of p‐InP.     

Nck-1 antagonizes the endoplasmic reticulum stress-induced inhibition of translation

Eukaryotic cells have developed specific mechanisms to overcome environmental stress. Here we show that the Src homology 2/3 (SH2/SH3) domain-containing protein Nck-1 prevents the unfolded protein response normally induced by pharmacological endoplasmic reticulum (ER) stress agents. Overexpression of Nck-1 enhances protein translation, whereas it abrogates eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and inhibition of translation in response to tunicamycin or thapsigargin treatment. Nck-1 overexpression also attenuates induction of the ER chaperone, the immunoglobulin heavy chain-binding protein (BiP), and impairs cell survival in response to thapsigargin. We provided evidence that in these conditions, the effects of Nck on the unfolded protein response (UPR) involve its second SH3 domain and a calyculin A-sensitive phosphatase activity. In addition, we demonstrated that protein translation is reduced in mouse embryonic fibroblasts lacking both Nck isoforms and is enhanced in similar cells expressing high levels of Nck-1. In these various mouse embryonic fibroblasts, we also provided evidence that Nck modulates the activation of the ER resident eIF2alpha kinase PERK and consequently the phosphorylation of eIF2alpha on Ser-51 in response to stress. Our study establishes that Nck is required for optimal protein translation and demonstrates that, in addition to its adaptor function in mediating signaling from the plasma membrane, Nck also mediates signaling from the ER membrane compartment.     

Characterization of Bacillus probiotics available for human use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.