The influence of orthography on phonemic knowledge: An experimental investigation on German and Persian

This study investigated whether the phonological representation of a word is modulated by its orthographic representation in case of a mismatch between the two representations. Such a mismatch is found in Persian, where short vowels are represented phonemically but not orthographically. Persian adult literates, Persian adult illiterates, and German adult literates were presented with two auditory tasks, an AX-discrimination task and a reversal task. We assumed that if orthographic representations influence phonological representations, Persian literates should perform worse than Persian illiterates or German literates on items with short vowels in these tasks. The results of the discrimination tasks showed that Persian literates and illiterates as well as German literates were approximately equally competent in discriminating short vowels in Persian words and pseudowords. Persian literates did not well discriminate German words containing phonemes that differed only in vowel length. German literates performed relatively poorly in discriminating German homographic words that differed only in vowel length. Persian illiterates were unable to perform the reversal task in Persian. The results of the other two participant groups in the reversal task showed the predicted poorer performance of Persian literates on Persian items containing short vowels compared to items containing long vowels only. German literates did not show this effect in German. Our results suggest two distinct effects of orthography on phonemic representations: whereas the lack of orthographic representations seems to affect phonemic awareness, homography seems to affect the discriminability of phonemic representations

Implementing Industry 4.0: Exploring the literature in a systematic way using text mining

The increasing popularity of digitisation practices and methods by scholars and practitioners alike has been paving the way for industrial transformation. Industry 4.0 has become an accepted trend across various industries, yet despite the increasing number of articles on this topic the complexities of implementation at the firm level remains largely vague and undefined. Therefore, the research presents a review of the social, operational and strategic aspects following the full-text mining of 116 selected articles. The study reveals that digital transformation requires stakeholders and investors to consider implementation through a multi-level and multidisciplinary lens. On this basis the study identifies the social, operational and strategic gaps within the literature and provides recommendations for future studies on implementation

Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

The electronic version of this article is the complete one and can be found online at: http://www.rbej.com/content/7/1/67Background: Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. Methods: Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48 h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. Results: Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. Conclusion: We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.Sandra Danner, Christiane Kirchhoff and Richard Ivel

Modelling germ cell development in vitro

Germ cells have a critical role in mediating the generation of genetic diversity and transmitting this information across generations. Furthermore, gametogenesis is unique as a developmental process in that it generates highly-specialized haploid gametes from diploid precursor stem cells through meiosis. Despite the importance of this process, progress in elucidating the molecular mechanisms underpinning mammalian germ cell development has been retarded by the lack of an efficient and reproducible system of in vitro culture for the expansion and trans-meiotic differentiation of germline cells. The dearth of such a culture system has rendered the study of germ cell biology refractory to the application of new high-throughput technologies such as RNA interference, leaving in vivo gene-targeting approaches as the only option to determine the function of genes believed to be involved in gametogenesis. Recent reports detailing the derivation of gametes in vitro from stem cells may provide the first steps in developing new tools to solve this problem. This review considers the developments made in modelling germ cell development using stem cells, and some of the challenges that need to be overcome to make this a useful tool for studying gametogenesis and to realize any future clinical application

Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

BACKGROUND: Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs). RESULTS: We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide. CONCLUSION: Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells. REVIEWERS: This article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter

Advanced immunostaining approaches to study early male germ cell development

Mammalian male germ cell development takes place in the testis under the influence of a variety of somatic cells and an incompletely defined paracrine and endocrine influences. Since it is not recapitulated well in vitro, researchers studying spermatogenesis often manipulate the germline by creating transgenic or knockout mice or by administering pharmaceutical agonists/antagonists or inhibitors. The effects of these types of manipulations on germline development can often be determined following microscopic imaging, both of stained and immunostained testis sections. Here, we describe approaches for microscopic analysis of the developing male germline, provide detailed protocols for a variety of immunostaining approaches, and discuss transgenic fluorescent reporter lines for studying the early stages of spermatogenesis

Primordial Germ Cell Specification from Embryonic Stem Cells

Background: Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo. Methodology and Principal Findings: Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stellanegative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation

Mechanisms Underlying Interferon-γ-Induced Priming of Microglial Reactive Oxygen Species Production.

Microglial priming and enhanced reactivity to secondary insults cause substantial neuronal damage and are hallmarks of brain aging, traumatic brain injury and neurodegenerative diseases. It is, thus, of particular interest to identify mechanisms involved in microglial priming. Here, we demonstrate that priming of microglia with interferon-γ (IFN γ) substantially enhanced production of reactive oxygen species (ROS) following stimulation of microglia with ATP. Priming of microglial ROS production was substantially reduced by inhibition of p38 MAPK activity with SB203580, by increases in intracellular glutathione levels with N-Acetyl-L-cysteine, by blockade of NADPH oxidase subunit NOX2 activity with gp91ds-tat or by inhibition of nitric oxide production with L-NAME. Together, our data indicate that priming of microglial ROS production involves reduction of intracellular glutathione levels, upregulation of NADPH oxidase subunit NOX2 and increases in nitric oxide production, and suggest that these simultaneously occurring processes result in enhanced production of neurotoxic peroxynitrite. Furthermore, IFNγ-induced priming of microglial ROS production was reduced upon blockade of Kir2.1 inward rectifier K+ channels with ML133. Inhibitory effects of ML133 on microglial priming were mediated via regulation of intracellular glutathione levels and nitric oxide production. These data suggest that microglial Kir2.1 channels may represent novel therapeutic targets to inhibit excessive ROS production by primed microglia in brain pathology

Multiple molecular mechanisms form a positive feedback loop driving amyloid β42 peptide-induced neurotoxicity via activation of the TRPM2 channel in hippocampal neurons

Emerging evidence supports an important role for the ROS-sensitive TRPM2 channel in mediating age-related cognitive impairment in Alzheimer’s disease (AD), particularly neurotoxicity resulting from generation of excessive neurotoxic Aβ peptides. Here we examined the elusive mechanisms by which Aβ₄₂ activates the TRPM2 channel to induce neurotoxicity in mouse hippocampal neurons. Aβ₄₂-induced neurotoxicity was ablated by genetic knockout (TRPM2-KO) and attenuated by inhibition of the TRPM2 channel activity or activation through PARP-1. Aβ₄₂-induced neurotoxicity was also inhibited by treatment with TPEN used as a Zn²⁺-specific chelator. Cell imaging revealed that Aβ₄₂-induced lysosomal dysfunction, cytosolic Zn²⁺ increase, mitochondrial Zn²⁺ accumulation, loss of mitochondrial function, and mitochondrial generation of ROS. These effects were suppressed by TRPM2-KO, inhibition of TRPM2 or PARP-1, or treatment with TPEN. Bafilomycin-induced lysosomal dysfunction also resulted in TRPM2-dependent cytosolic Zn²⁺ increase, mitochondrial Zn²⁺ accumulation, and mitochondrial generation of ROS, supporting that lysosomal dysfunction and accompanying Zn²⁺ release trigger mitochondrial Zn²⁺ accumulation and generation of ROS. Aβ₄₂-induced effects on lysosomal and mitochondrial functions besides neurotoxicity were also suppressed by inhibition of PKC and NOX. Furthermore, Aβ₄₂-induced neurotoxicity was prevented by inhibition of MEK/ERK. Therefore, our study reveals multiple molecular mechanisms, including PKC/NOX-mediated generation of ROS, activation of MEK/ERK and PARP-1, lysosomal dysfunction and Zn²⁺ release, mitochondrial Zn²⁺ accumulation, loss of mitochondrial function, and mitochondrial generation of ROS, are critically engaged in forming a positive feedback loop that drives Aβ₄₂-induced activation of the TRPM2 channel and neurotoxicity in hippocampal neurons. These findings shed novel and mechanistic insights into AD pathogenesis